Methods for reprogramming and gene editing cells

Abstract

The present disclosure provides improved methods for reprogramming and gene editing cells, including manufacturing a population of cells comprising cells of the lymphoid lineage and / or cells of the myeloid lineage.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a National Stage Entry of International Application No. PCT / US2023 / 066464, which claims the benefit of U.S. Provisional Patent Application No. 63 / 337,166, filed May 1, 2022, and U.S. Provisional Patent Application No. 63 / 342,144, filed May 15, 2022, each of which is entirely incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on May 1, 2023, is named 61057-719_601.xml and is 81,727 bytes in size.BACKGROUND

[0003] Methods for reprogramming differentiated cells into pluripotent cells and methods for gene-editing cells have progressed greatly over recent years. However, there remain unmet needs for improved methods for reprogramming and gene editing cells.SUMMARY

[0004] Accordingly, the present disclosure provides improved methods for reprogramming and gene editing cells.

[0005] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0006] In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR), e.g., a CAR which comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In some cases, the antigen binding region binds to one or more tumor antigens. In various cases, the CAR comprises an antigen binding region that binds to ROR1. In embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine. In various embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein and a single-stranded or double-stranded repair template which encodes a chimeric antigen receptor (CAR). In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the CAR inserts into the break. In these embodiments, the cell in the subject expresses the CAR.

[0007] In numerous embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein and a single-stranded or double-stranded repair template which encodes a cytokine. In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the cytokine inserts into the break. In these embodiments, the cell in the subject expresses or over expresses the cytokine.

[0008] In various embodiments, the isolated lymphoid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes; wherein steps (5) and (6) occur in an adherent culturing vessel.

[0009] In embodiments, the isolated myeloid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a myeloid progenitor medium; and (6) culturing the cells of step (5) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5) and (6) occur in a bioreactor.

[0010] Another aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for treating a cancer.

[0011] Yet another aspect of the present disclosure is a method for killing a cancer cell or for inhibiting the proliferation of a cancer cell. The method comprising contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0012] In some embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vitro.

[0013] In various embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vivo.

[0014] Yet a further aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for killing a cancer cell or for inhibiting the proliferation of a cancer cell.

[0015] In an aspect, the present disclosure provides a method for manufacturing a plurality of population of cells comprising a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells for treating a cancer, for killing a cancer cell, and / or for inhibiting the proliferation of a cancer cell. The method a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor.

[0016] An aspect of the present disclosure is a method for manufacturing a population of cells that is enriched for cytotoxic lymphocytes. The method comprises steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes. In this aspect, steps (5) and (6) occur in an adherent culturing vessel. When CD34+ cells are selected, the embryoid bodies may be first chemically and / or mechanically dissociated.

[0017] Another aspect of the present disclosure is a method for killing a cancer cell. The method comprising steps of: (1) obtaining a herein-disclosed cytotoxic lymphocyte and (2) contacting cytotoxic lymphocyte with the cancer. In some cases, the cancer cell is in vivo.

[0018] Yet another aspect of the present disclosure is a method for treating a cancer patient in need thereof. The method comprising a step of administering to the cancer patient a therapeutically-effective amounts of a herein-disclosed cytotoxic lymphocyte.

[0019] In an aspect, the present disclosure provides a pharmaceutical composition comprising a herein-disclosed cytotoxic lymphocyte and a pharmaceutically acceptable carrier or excipient.

[0020] In another aspect, the present disclosure provides a composition comprising a cell comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, wherein the cell is a cytotoxic lymphocyte from a lymphoid lineage cell, e.g., an NK cell, or the cell is from a myeloid lineage, e.g., a macrophage, or the cell is a mesenchymal stromal / stem cell, or the cell is a hematopoietic stem cell.

[0021] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated NK cell of any herein-disclosed aspect or embodiment.

[0022] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated myeloid cell of any herein-disclosed aspect or embodiment, e.g., a macrophage.

[0023] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated mesenchymal stromal / stem cell of any herein-disclosed aspect or embodiment.

[0024] An aspect of the present disclosure is a method of making an engineered cell comprising a disruption in a beta-2-microglobulin (B2M) gene. The method comprising steps of (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins; and (c) differentiating the iPS cell into a differentiated cell. In this aspect, the differentiated cell is a cytotoxic lymphocyte from a lymphoid cell lineage or is from a myeloid cell lineage, e.g., a macrophage.

[0025] Another aspect of the present disclosure is a method of treating cancer. The method comprising steps of obtaining an isolated cell comprising a genetically engineered disruption in a B2M gene and administering the isolated cell to a subject in need thereof. In this aspect, the cytotoxic lymphocyte is a lymphoid cell or a CAR-myeloid cell or a CAR-mesenchymal stromal / stem cell.

[0026] Yet another aspect of the present disclosure is a composition comprising an isolated cytotoxic lymphocyte comprising a gene edit in a CD16a gene, wherein the cytotoxic lymphocyte is a lymphoid lineage cell, e.g., an NK cell.

[0027] An aspect of the present disclosure is a method for producing macrophages from an induced a pluripotent stem cell (iPSC). The method comprises steps of: (1) obtaining an iPSC; (2) culturing the iPSC in a first medium for about three days; (3) culturing the iPSC in a second for about four days; (4) culturing the iPSC in a monocyte differentiating medium for at least seven days, thereby obtaining monocytes (5) isolating the monocytes; (6) culturing the monocytes for about four days; (7) culturing the monocytes in the presence of M-CSF for three to four days, thereby obtaining macrophages; and (8) harvesting the macrophages. In some embodiments, the macrophages are further contacted with interferon gamma (IFN-γ) and / or lipopolysaccharide (LPS) to obtain M1 macrophages and / or the macrophages are further contacted with IL-4 to obtain M2 macrophages.

[0028] In embodiments, the macrophages, e.g., the M1 and M2 macrophages, are capable of killing cancer cells.

[0029] In embodiments, the iPSC was reprogrammed from a differentiated or non-pluripotent cell.

[0030] In embodiments, the iPSC or a progenitor cell was gene-edited. In some cases, the iPSC or the progenitor cell was gene-edited to knockout the beta-2 microglobulin (B2M) gene.

[0031] In embodiments, the gene-editing comprises transfection of a repair template.

[0032] In embodiments, the repair template includes the coding sequence for one or more of HLA class I histocompatibility antigen, alpha chains (HLAs).

[0033] In embodiments, the repair template comprises a TTAGGG motif for decreasing synthetic oligodeoxynucleotides (ODNs)-related activation of pro-inflammatory responses and / or the cell is transfected with a TTAGGG motif separate from the repair template.

[0034] In embodiments, the differentiated or non-pluripotent cell was contacted with resveratrol before reprogramming.

[0035] In embodiments, the iPSC was contacted with resveratrol before gene-editing and / or the iPSC was contacted with resveratrol after gene-editing.

[0036] Another aspect is an isolated macrophage obtained by a herein disclosed method.

[0037] Yet another aspect is pharmaceutical composition comprising a herein disclosed isolated macrophage of and a pharmaceutically-acceptable excipient.

[0038] In an aspect, the disclosure provides an isolated M1 macrophage and / or an isolated M2 macrophage obtained by a herein disclosed method.

[0039] In yet another aspect, the disclosure provides a pharmaceutical composition comprising a herein disclosed isolated M1 macrophage and / or an isolated M2 macrophage and a pharmaceutically-acceptable excipient.

[0040] In another aspect, the disclosure provides method for treating a cancer comprising in vivo administering to a subject in need a herein disclosed pharmaceutical composition.

[0041] An aspect of the present disclosure is a method for decreasing synthetic oligodeoxynucleotides (ODNs)-related activation of pro-inflammatory responses, the method comprising transfecting a cell with an ODN comprising a TTAGGG motif.

[0042] In embodiments, the ODN is a double stranded ODN (dsODN) and comprise a repair template.

[0043] In embodiments, the TTAGGG motif is attached to the 5′ and / or the 3′ end of the repair template.

[0044] In embodiments, the ODN is a single stranded ODN (ssODN) and does not comprise a repair template.

[0045] In embodiments, the cell is transfected with a synthetic nucleic acid encoding a gene-editing protein along with a repair template.

[0046] Another aspect is an isolated cell obtained by a method relating to an ODN comprising a TTAGGG motif.

[0047] Yet another aspect is a method for enhancing the efficiency of gene-editing, the method comprising contacting a cell with resveratrol before gene-editing.

[0048] In embodiments, contacting the cell with resveratrol arrests the cell in S / G2 phase.

[0049] In embodiments, the cell is further contacted with resveratrol after gene-editing.

[0050] In embodiments, gene-editing comprise transfection of a synthetic nucleic acid encoding a gene-editing protein.

[0051] In an aspect, the present disclosure provides a method for enhancing the efficiency of gene-editing, the method comprising contacting a cell that has been gene-edited with resveratrol.

[0052] In embodiments, the gene-editing comprise transfection of a synthetic nucleic acid encoding a gene-editing protein.

[0053] Any aspect or embodiment herein may be combined with any other aspect or embodiment as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1A shows a non-limiting schematic of the mRNA-based reprogramming and gene-editing, followed by differentiation of the present disclosure. FIG. 1B illustrates differentiated cells killing cancer cells.

[0055] FIG. 2 shows the design of the gene-editing scheme for beta-2-microglobulin (B2M); shown are the following sequences: TCATCCATCCGACATTGA (SEQ ID NO: 1), AGTTGACTTACTGAAG (SEQ ID NO: 2), AATGGAGAGAGAATTGAA (SEQ ID NO: 3).

[0056] FIG. 3 shows an RNA gel demonstrating gene-editing of B2M.

[0057] FIG. 4 shows a sequencing experiment that shows the 14 base pair deletion from a gene-edited B2M; shown are the following sequences from bottom to top: ACATTGAAGAATGGAG (SEQ ID NO: 4), ACATTGAAGTTGACTTACTGAAGAATGGAG (SEQ ID NO: 5), and TGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAAT GGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGT (SEQ ID NO: 6).

[0058] FIG. 5 shows RNA levels of B2M with or without IFN gamma activation (“IFNY”; two left bars are the B2M knockout, and the two right bars are naïve cells).

[0059] FIG. 6 shows a sequencing experiment that demonstrates heterozygosity of CD16a (at G147D dbSNP:rs443082, Y158H dbSNP:rs396716, and F176V dbSNP:rs396991); shown are the following sequences from top to bottom: GKGRKYFHHNSDFHIPKATLKDS (SEQ ID NO: 7), GKDRKYFHHNSDFYIPKATLKDS (SEQ ID NO: 8), KDSGSYFCRGLFGSKNVSSETVN (SEQ ID NO: 9), and KDSGSYFCRGLVGSKNVSSETVN (SEQ ID NO: 10).

[0060] FIG. 7A-7B shows images of control (PMBC-isolated) NK cells in co-culture with K-562 tumor cells, demonstrating NK Cell cytotoxicity of tumor cell (note immunothrombosis or “clumping”).

[0061] FIG. 8A-8B shows images of the gene edited and differentiated cells of the present disclosure (e.g., B2M knockout NK cells) in co-culture with K-562 tumor cells, demonstrating NK Cell cytotoxicity of tumor cell (note immunothrombosis or “clumping”).

[0062] FIG. 9A-FIG. 9H show results of the cytokine release assay with the Luminex MAGPIX. Unless indicated (i.e., “+IL2, IL15”), conditions are without added IL-2 or IL-15. Further, ratio of cells is indicated (1:1 or 3:1). As elsewhere herein, PBMC-NK are control NK cells. FIG. 9A shows interferon gamma. FIG. 9B shows IL-2. FIG. 9C shows IL-7. FIG. 9D shows IL-13. FIG. 9E shows MIP-1a. FIG. 9F shows MIP-1b. FIG. 9G shows TNFα. FIG. 9H shows GM-CSF.

[0063] FIG. 10A-FIG. 10D show flow cytometry data for a gene edited and differentiated cells of the present disclosure (e.g., B2M knockout NK cells) as described in the Examples.

[0064] FIG. 11A shows the structure for the B2M-HLA-E repair template. FIG. 11B shows an ideal target site for the B2M-HLA-A repair template is shown (SEQ ID NO: 11: MSRSVALAVLALLSLSGLEAIQ; and SEQ ID NO: 12 ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGC TATCCAGCgtgagtctctcctaccctcccgctc). FIG. 11C shows additional target binding sites (SEQ ID NO: 11 and SEQ ID NO: 12 are again shown). FIG. 11D shows a gel with sizes of two lines having the B2M-HLA-E repair template inserted. FIG. 11E includes graphs showing the intensities of signal and ratios thereof from the bands shown in FIG. 11D. FIG. 11F shows a gel with sizes of two lines having the B2M-HLA-E repair template inserted. FIG. 11G includes graphs showing the intensities of signal and ratios thereof from the bands shown in FIG. 11F. FIG. 11H shows relevant sequences in the B2M-HLA-E repair template.

[0065] FIG. 12A and FIG. 12B, show target site sequences and repair templates for replacing the phenylalanine (F) at position 158 of CD16a with a valine (V). Relevant sequences are shown in these figures.

[0066] FIG. 13 is a graphical representation of different protocols in the differentiation of cytotoxic lymphocytes.

[0067] FIG. 14 is an illustrative flow cytometry fluorescence map used in data analysis of cytotoxicity assays.

[0068] FIG. 15 are graphs showing percentages of cancer killed in 24 hours. The left data for each graph are cells that were not activated and the right data for each graph are cells that were activated with IL-15 and IL-2.

[0069] FIG. 16 are graphs showing the ability of cytotoxic lymphocytes to kill K692 cancer cells and their inability to kill NK-resistant cancer cells.

[0070] FIG. 17 is a scatter plot showing two distinct populations of cells.

[0071] FIG. 18 are scatter plots for cells derived from Protocol 2, 3, or 4 as illustrated in FIG. 13.

[0072] FIG. 19 is a cartoon showing methods for manufacturing mixed population iPS-cell derived lymphoid lineage cells and myeloid lineage cells for enhanced tumor cell killing.

[0073] FIG. 20 shows an overview of scalable iPSC differentiation into lymphoid and myeloid cells.

[0074] FIG. 21 includes photomicrographs showing morphology of iPSC to macrophage progenitor cells in the T75 and Bioreactor.

[0075] FIG. 22 includes graphs showing the sum of the viable macrophage progenitor cells harvested from the T75 (left) and bioreactor (right) throughout the culture period. * Indicates when a large harvest (>50% of total cells) was performed.

[0076] FIG. 23 is a graph assessing baseline macrophage cytotoxicity.

[0077] FIG. 24 includes florescent photomicrographs of mRNA transfection of macrophages.

[0078] FIG. 25 includes photomicrographs showing morphology of NK cells on the final day of the differentiation protocol (left) versus 24 hours post thaw (right).

[0079] FIG. 26 is a graph showing the effects of cryopreservation on the cytotoxicity of NK cells.

[0080] FIG. 27 includes a graph (left) and photomicrographs (right) of data from an isolated huPBMC mixed cell type cytotoxicity assay.

[0081] FIG. 28 includes data from cytotoxicity assay cytokine release heat maps with iPS derived macrophages and NK cells. * Indicates below level of detection. ** Indicates sample read N / A.

[0082] FIG. 29 includes photomicrographs showing iPSC derived immune cell clustering during a cytotoxicity assay after 24 hours.

[0083] FIG. 30 is a cartoon showing methods for rapid prototyping of macrophage gene-editing strategies for cancer immunotherapies.

[0084] FIG. 31 is a timeline showing efficient differentiation of iPSCs into macrophages.

[0085] FIG. 32 is a photomicrograph showing iPSC-macrophages displaying macrophage-like morphology following differentiation.

[0086] FIG. 33 is a schematic of an mRNA encoding a ROR1-CAR.

[0087] FIG. 34A includes florescent photomicrographs and FIG. 34B includes graphs of data from GFP-encoding mRNA transfected into macrophages.

[0088] FIG. 35A includes florescent photomicrographs and FIG. 35B includes graphs of data from macrophages transfected with mRNA encoding an ROR1-CAR.

[0089] FIG. 36 includes photomicrographs of data from a Zymosan bead phagocytosis assay.

[0090] FIG. 37 includes photomicrographs of data from a CD3ζ phosphorylation assay.

[0091] FIG. 38 includes photomicrographs (left) and a graph (right) of data from an SKOV3 cytotoxicity assay.

[0092] FIG. 39 is a cartoon showing a protocol for insertion of transgene.

[0093] FIG. 40 is a schematic of the structure of a ROR1-CAR transgene.

[0094] FIG. 41 is a gel showing insertion of a ROR1-CAR transgene into iPSC lines.

[0095] FIG. 42 is a cartoon showing methods for reprogramming fibroblasts into induced pluripotent stem cells (iPSCs), which are then differentiated into monocytes which are further differentiated into cancer killing macrophages.

[0096] FIG. 43A to FIG. 43C show general steps for the process of differentiating iPSCs to macrophage.

[0097] FIG. 43A shows iPSC to monocyte differentiation, FIG. 43B shows CD14+ magnetic bead positive selection, and FIG. 43C shows monocyte to macrophage differentiation.

[0098] FIG. 44 shows progressing of cells from an iPSC colony (top left), on day 3 mesoderm (top right), on day 7 hematopoietic stem cell (bottom left), and on day 14 monocyte (bottom right).

[0099] FIG. 45A to FIG. 45C are flow cytograms showing peaks marked by CD14 (FIG. 45A), CD45 (FIG. 45B), and CD163 (FIG. 45C) for cryopreserved PBMC-monocytes and for cryopreserved iPSC-monocytes flowed directly after thawing.

[0100] FIG. 46 shows monocyte cultures at day zero (left image) and at day 4 and upon activation with M-CSF (right image).

[0101] FIG. 47 shows macrophage cultures at day zero (left image) and at day 3 after (right image).

[0102] FIG. 48 is a graph showing ELISA on iPSC-Macrophage supernatants (1M cells / mL).

[0103] FIG. 49 shows the process for testing the cancer-cell killing ability of iPSC-macrophages of the present disclosure.

[0104] FIG. 50 is a flow cytometry scatter plot showing iPSC-derived macrophages killing of U2OS cancer cells in vitro.

[0105] FIG. 51 is a chart showing that iPSC-derived macrophages killed 45% of U2OS cancer cells in vitro.

[0106] FIG. 52 is a flow cytometry scatter plot showing iPSC-derived macrophages killing of MA011sk cells or donor fibroblasts in vitro.

[0107] FIG. 53 is a chart showing that iPSC-derived macrophages do not kill MA011sk cells or donor fibroblasts in vitro.

[0108] FIG. 54 is a cartoon showing methods for reducing an immune response by including a TTAGGG motif in an dsODN.

[0109] FIG. 55A to FIG. 55C shows that iMSC electroporated with a synthetic nucleic acid encoding a gene-editing protein and with a repair template comprising the code for GFP, expressed GFP 24 hours after electroporation (FIG. 55A), 72 hours after electroporation (FIG. 55B), and 28 days hours after electroporation and by passage 4 (FIG. 55C).

[0110] FIG. 56 is a blot showing results from gene-editing iMSCs with the gene-editing protein alone (lane 3) or with the gene-editing protein, the A151 oligo, dsODN repair template, and U11 (lane 4).

[0111] FIG. 57 are flow cytometry scatter plots for iMSCs that were gene edited along with the A151 ODN.

[0112] FIG. 58 is a cartoon showing the process of contacting Resveratrol with a cell in advance of gene editing.

[0113] FIG. 59 are graphs showing numbers of cells in S / G2 that were not pretreated with Resveratrol (left graph) and numbers of cells in S / G2 that were pretreated with Resveratrol (right graph).

[0114] FIG. 60 is a gel showing that Resveratrol pretreated fibroblasts have increased gene-editing efficiency.

[0115] FIG. 61 is a gel showing that that Resveratrol treatment after electroporation with gene-editing nucleic acids increased 1 kb insertion 1.6-fold in iPSC.

[0116] FIG. 62 is a gel showing that NU7441 (a DNA-PKs inhibitor that is known to inhibit NHEJ mediated DNA repair pathway) have increased gene-editing efficiency.

[0117] FIG. 63 is a gel showing that a maximum 32% of 1 kb insertion rate was observed using ssDNA repair template in iPSCs.DETAILED DESCRIPTIONMethods for Manufacturing Cytotoxic Lymphocytes

[0118] An aspect of the present disclosure is a method for manufacturing a population of cells that is enriched for cytotoxic lymphocytes. The method comprises steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes. In this aspect, steps (5) and (6) occur in an adherent culturing vessel. When CD34+ cells are selected, the embryoid bodies may be first chemically and / or mechanically dissociated.

[0119] In embodiments, the stem cell is an induced pluripotent stem (iPSC).

[0120] In some embodiments, the stem cell has a wild-type genome or has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene. In some cases, the stem cell has a biallelic disruption in a B2M gene.

[0121] In some cases, mRNA-reprogrammed iPSC lines with a biallelic knockouts of the beta-2 microglobulin (B2M) gene, a key component of MHC class I molecules, are obtained using an mRNA-encoded chromatin context-sensitive gene-editing endonuclease. The B2M-knockout iPSCs may be differentiated using a novel, fully suspension process that replaces specialized micropatterned culture vessels with a spheroid culture step. Additional details regarding B2M knockout iPSCs useful in the present disclosure are described in PCT / US2022 / 019020, the contents of which are incorporated herein by reference in its entirety.

[0122] In various embodiments, the bioreactor is suited for culturing shear-sensitive cells and / or does not require use of anti-foaming agents or shear protectants, e.g., a vertical wheel bioreactor such as a PBS Biotech vertical-wheel bioreactor.

[0123] In embodiments, the medium in step (2) is serum-free and feeder-free culture medium, e.g., an mTeSR™ medium.

[0124] In some embodiments, the medium in step (6) is a serum-free and feeder-free culture medium, e.g., a StemDiff™ NK medium.

[0125] In various embodiments, the adherent culturing vessel is a multi-well plate or a cell culturing flask.

[0126] In embodiments, the method provides from about 10-fold to about 100-fold more cytotoxic lymphocytes than obtained by a method in which each of the culturing steps comprise adherent culturing vessels; obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels; and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0127] In some embodiments, the cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells.

[0128] In various embodiments, the method provides from about 5-fold to about 30-fold more CD16+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0129] In embodiments, the method provides from about 5-fold to about 25-fold more NDG2D+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0130] In some embodiments, the method provides from about 2-fold to about 30-fold more NKp44+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0131] In various embodiments, the method provides from about 2-fold to about 8-fold more CD94+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0132] In embodiments, the method provides from about 2-fold more NKp46 cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels.

[0133] In some embodiments, the cytotoxic lymphocyte targets and kills cancer cells, e.g., a K562 cancer cell. In various embodiments, the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. In embodiments, the cytotoxic lymphocyte targets and kills at least 70% of cancer cells in a population within about 4 hours. In some embodiments, the cytotoxic lymphocyte targets and kills at least 80% of cancer cells in a population within about 24 hours.

[0134] In various embodiments, the cytotoxic lymphocyte has reduced cytotoxicity to an NK-resistant cancer cell, e.g., a NAMALWA cell.

[0135] In embodiments, the cytotoxic lymphocyte is a Natural Killer (NK) cell. In some cases, the NK cell is a mature NK cell.

[0136] In some embodiments, the cytotoxic lymphocyte is a Natural killer T (NKT) cell.

[0137] In various embodiments, the cytotoxic lymphocyte is a delta-gamma T cell.

[0138] In embodiments, the iPSC was reprogrammed from a somatic cell comprising contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors.Cytotoxic Lymphocytes

[0139] In embodiments, the present cytotoxic lymphocyte is of the lymphoid cell lineage or the myeloid cell lineage.

[0140] In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell.

[0141] In some cases, the lymphoid cell is an NK cell, e.g., an NK-T cell. The NK cell may be a human cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0142] In various embodiment, the cytotoxic lymphocyte is reprogrammed from a stem cell, e.g., an iPSC, and differentiated into the cytotoxic lymphocyte.

[0143] In embodiments, the cytotoxic lymphocyte has a disruption in its beta-2-microglobulin (B2M) gene. In embodiments, the cytotoxic lymphocyte has a disruption in its beta-2-microglobulin (B2M) gene and expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0144] In embodiments, the cytotoxic lymphocyte is gene edited to express a high affinity variant of CD16a (See, FIG. 12A and FIG. 12B).

[0145] In embodiments, the myeloid lineage cell is a cell derived from, or derivable from, a common myeloid progenitor cell. In embodiments, the myeloid cell is a megakaryocyte, erythrocyte, mast cell, or myeloblast. In embodiments, the myeloid cell is a cell derived from, or derivable from, a myeloblast. In embodiments, the myeloid cell is a basophil, neutrophil, eosinophil, or monocyte. In embodiments, the myeloid cell is a cell derived from, or derivable from a monocyte. In embodiments, the myeloid cell is a macrophage. In embodiments, the myeloid cell is a dendritic cell.

[0146] In embodiments, the cytotoxic lymphocyte is an NK cell. In embodiments, the NK cell is a human cell. In embodiments, the NK cell is derived from somatic cell of a subject. In embodiments, the NK cell is derived from allogeneic or autologous cells. In embodiments, the NK cell is derived from an induced pluripotent stem (iPS) cell. In embodiments, the iPS is derived from reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors, optionally selected from Oct4, Sox2, cMyc, and Klf4. In embodiments, the iPS cell is derived from allogeneic or autologous cells. In embodiments, the NK cell expresses one or more of CD56 and CD16.

[0147] In embodiments, the NK cell expresses CD16a, which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V (See, FIG. 12A and FIG. 12B).

[0148] In embodiments, the NK cell does not express CD3.

[0149] In embodiments, the NK cell is CD56bright CD16dim / −. In embodiments, the NK cell is CD56dim CD16+. In embodiments, the NK cell is a NKtolerant cell, optionally comprising CD56bright NK cells or CD27− CD11b− NK cells. In embodiments, the NK cell is a NKcytotoxic, optionally comprising CD56dim NK cells or CD11b+ CD27− NK cells. In embodiments, the NK cell is a NKregulatory, optionally comprising CD56bright NK cells or CD27+ NK cells. In embodiments, the NK cell is a natural killer T (NKT) cell. In embodiments, the NK cell secretes one or more cytokines selected from interferon-gamma (IFN-g), tumor necrosis factor-alpha (TNF-a), tumor necrosis factor-beta (TNF-b), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b).

[0150] In embodiments, the present cytotoxic lymphocyte has reduced or eliminated cytotoxic lymphocyte fratricide, e.g., NK-cell fratricide. For instance, in embodiments, the present engineered NK cells surprisingly do not engage in NK cytotoxicity and therefore are able to survive despite disruptions, e.g., in beta-2-microglobulin (B2M).

[0151] In embodiments, the present cytotoxic lymphocyte is capable of self-activating. In embodiments, the present cytotoxic lymphocyte is capable of activating without the need for extracellular signals (e.g., cytokines), including signals that may be provided exogenously. In embodiments, the present cytotoxic lymphocyte does not require ex vivo stimulation for activity. In embodiments, the present cytotoxic lymphocyte is capable of self-activating in the absence of an interleukin, optionally selected from IL-2 and IL-15.

[0152] In embodiments, the present cytotoxic lymphocyte is capable of inducing tumor cell cytotoxicity. In embodiments, the present cytotoxic lymphocyte is capable of inducing tumor cell cytotoxicity in the absence of an interleukin, optionally selected from IL-2 and IL-15. Assays for assessing tumor cell cytotoxicity include in vivo anti-cancer response evaluation, as well as microscopic evaluation, e.g., a calcein acetoxymethyl (AM) staining-based microscopic method (See EXAMPLES and Chava et al. J Vis Exp. 2020 Feb. 22; (156): 10.3791 / 60714, the entire contents of which are incorporated by reference). Further, a colorimetric lactic dehydrogenase (LDH) measurement-based NK cell-mediated cytotoxicity assay may be employed (see Chava et al. J Vis Exp. 2020 Feb. 22; (156): 10.3791 / 60714, the entire contents of which are incorporated by reference).Scalable, Mixed Population iPS-Cell Derived Cytotoxic Lymphocytes and Myeloid Cells

[0153] Induced pluripotent stem cell (iPSC) therapies have the potential to treat a wide variety of devastating diseases. iPSC-derived lymphocytes (e.g., T cells and NK cells) engineered to express targeting molecules such as chimeric antigen receptors (CARs) have shown clinical promise to treat hematological malignancies. More recently, iPS cell-derived myeloid cells are being developed to treat both hematological malignancies and solid tumors due to the ability of these cells to infiltrate and modulate the tumor microenvironment. Despite preliminary success, several challenges still remain, including poor infiltration of cytotoxic lymphocytes into solid tumors and insufficient cytotoxicity of myeloid cells.

[0154] As is known in the art, an animal's immune system comprises a wide variety of immune cell types capable of contributing to an anti-cancer effect. And, in vivo, one type of immune cell promotes the cancer-killing ability of a second type of immune cell. Notably, NK cells are expert in killing cancer cells but rarely not infiltrate solid tumors alone and require recruitment by macrophages which have already infiltrated the solid tumor and, on the other hand, macrophages are less adept at killing cancer cells but expert in infiltrating solid tumors and secreting cytokines that recruit cancer killing cells. Thus, each type of immune cell has its function which work in cooperation with the other cell types to attack and kill cancer cells. Nonetheless, many cell-based cancer therapeutics in clinical trials employ one type of immune cell rather than a plurality of immune cell types as existent in vivo. Without wishing to be bound by theory, a multi-cell-type therapy comprising both lymphocyte and myeloid cells may work synergistically, enhancing cytotoxicity and efficacy.

[0155] This disclosure, e.g., in Examples 8 and 9, describes a scalable bioreactor-based process for parallel differentiation of mRNA reprogrammed iPSC into both CD14+ (>95% positive) macrophages and CD56bright / CD16dim NK cells. This process yielded 1×106 myeloid cells / ml and 3×105 lymphoid cells / ml, and is amenable to scaling to clinically relevant doses. In vitro, the lymphoid and myeloid cells showed synergistic tumor cell killing of SKOV3 ovarian cancer cells (combined: 15.6%; macrophage alone=2.2% (p<0.01); NK alone=7.5% (p<0.05); E:T=5:1). The combined cells showed increased expression of TNFα and demonstrated enhanced clustering and tumor cell engagement. To further improve the macrophages' ability to target and infiltrate solid tumors, macrophages were transfected with mRNA encoding a humanized ROR1-CAR protein. mRNA transfection increased cytotoxicity towards SKOV3 cells by 6-fold.

[0156] In conclusion, the present disclosure provides a scalable platform for generating iPSC-derived multi-cell-type therapies comprising both lymphoid and myeloid cells. These cells act synergistically to kill tumor cells in vitro. And, by closely mimicking natural cellular immunity, multi-cell-type cell therapies represent a new class of cell therapies that may play an important role in the development of new medicines for treating cancer.

[0157] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0158] In embodiments, the first pharmaceutical composition comprises the population of isolated lymphoid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated myeloid lineage cells.

[0159] In some embodiments, the first pharmaceutical composition comprises the population of isolated myeloid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated lymphoid lineage cells. In some cases, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or sequentially. The first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the first pharmaceutical composition administered before the second pharmaceutical composition or the first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the second pharmaceutical composition administered before the first pharmaceutical composition.

[0160] In various embodiments, the first pharmaceutical composition comprises both the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells.

[0161] In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR), e.g., a CAR which comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In some cases, the antigen binding region binds to one or more tumor antigens. In various cases, the CAR comprises an antigen binding region that binds to ROR1. In embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine. In some embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, optionally, wherein the cells express a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0162] In various embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a chimeric antigen receptor (CAR). In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the CAR inserts into the break. In these embodiments, the cell in the subject expresses the CAR.

[0163] In numerous embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a cytokine. In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the cytokine inserts into the break. In these embodiments, the cell in the subject expresses or over expresses the cytokine.

[0164] When the synthetic mRNA and / or the repair template is administered to a subject, the synthetic mRNA and / or the repair is combined with a lipid system comprising a compound of Formula (IV).

[0165] In various cases, transfection of a cell with synthetic nucleic acids for gene-editing may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In embodiments, the cell in the subject (e.g., which expresses the CAR and / or cytokine) is of the lymphoid lineage or is of the myeloid lineage.

[0167] In some embodiments, the isolated lymphoid lineage cell and / or the isolated myeloid lineage cell is derived from an induced pluripotent stem cell (iPSC). In some cases, the isolated lymphoid lineage cell and the isolated myeloid lineage cell is derived from the same iPSC. The iPSC comprises a genomic modification that expresses a chimeric antigen receptor (CAR) and / or the iPSC comprises a genomic modification that expresses or over expresses a cytokine. In various cases, the iPSC comprises a genomic modification which disrupts the beta-2-microglobulin (B2M) gene.

[0168] In various embodiments, the isolated lymphoid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes; wherein steps (5) and (6) occur in an adherent culturing vessel.

[0169] In numerous embodiments, the isolated lymphoid lineage cells comprise cytotoxic lymphocytes. In some cases, the isolated lymphoid lineage cells comprising cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells. In these embodiments, the cytotoxic lymphocyte targets and kills cancer cells and, in some cases, the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. The cytotoxic lymphocyte has reduced cytotoxicity to an NK-resistant cancer cell. In various cases, the cytotoxic lymphocyte is a Natural Killer (NK) cell, e.g., a mature NK cell, or is a cytotoxic T cell. In these embodiments, the cytotoxic lymphocyte is a Natural killer T (NKT) cell. In some cases, the NK cell expresses CD16a and / or the NK cell does not express CD3, and / or the NK cell is CD56bright CD16dim / −. In many cases, the NK cell secretes one or more cytokines selected from interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b). The cytotoxic lymphocyte may be a delta-gamma T cell. In numerous cases, the cytotoxic lymphocyte is further engineered to express a chimeric antigen receptor (CAR) and / or is further engineered to express or overexpress a cytokine.

[0170] In embodiments, the isolated myeloid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a myeloid progenitor medium; and (6) culturing the cells of step (5) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5) and (6) occur in a bioreactor.

[0171] In some embodiments, the isolated myeloid lineage cells comprise a megakaryocyte, erythrocyte, mast cell, myeloblast, dendritic cell, basophil, neutrophil, eosinophil, monocyte, or macrophage.

[0172] In various embodiments, the isolated myeloid lineage cells express one or more of CD11b, CD13, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., in amounts that are similar to amounts expressed by PBMC-derived cells.

[0173] In numerous embodiments, the isolated myeloid lineage cells have increased expression of CD80 and / or CD206, which is indicative of an activated state.

[0174] In embodiments, the isolated myeloid lineage cell is a macrophage. In some cases, the macrophage expresses one or more of CD11b, CD68, CD80, CD86, CD163, CD206, and SIRPα in amounts that are similar to amounts expressed by PBMC-derived cells and / or secretes one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells. In various cases, the macrophage expresses one or more of CD34, CD44, CD45, CD73, and CD90. In these embodiments, the method further comprises a step of differentiating the macrophages into M1 and / or M2 macrophages, e.g., by exposure to MCSF. And, the method may further comprise a step of polarizing the M1 macrophages with interferon gamma (IFN-γ) and / or lipopolysaccharide (LPS) and / or treating the M2 macrophages with IL-4. In these cases, the macrophages comprise M1 macrophages and / or M2 macrophages. The M1 macrophages and / or M2 macrophages secrete one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells.

[0175] In some embodiments, the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0176] In various embodiments, the isolated myeloid lineage cell is further engineered to express a chimeric antigen receptor (CAR).

[0177] In numerous embodiments, the isolated myeloid lineage cell is further engineered to express or overexpress a cytokine. In cases when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated.

[0178] In embodiments, the stem cell is an induced pluripotent stem (iPSC). In some cases, the stem cell stem has a wild-type genome or has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a biallelic disruption in a B2M gene. In various cases, the stem cell expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0179] In some embodiments, the iPSC was reprogrammed from a somatic cell and the method further comprises contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors. The one or more reprogramming factors may be selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In some cases, the somatic cell is selected from fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0180] In embodiments where the stem cell is an iPSC, the iPSC is further engineered to express a chimeric antigen receptor (CAR) and / or the iPSC is further engineered to express or overexpress a cytokine. In various embodiments, the isolated lymphoid lineage cells and the isolated myeloid lineage cells are manufactured by a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor. In cases when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated. In various cases, the stem cell is an induced pluripotent stem (iPSC).

[0181] In embodiments, the method of manufacturing provides at least 1×106 myeloid lineage cells / ml and at least 3×105 lymphoid lineage cells / ml.

[0182] In some embodiments, the method of manufacturing provides both CD14+ (>95% positive) macrophages and CD56bright / CD16dim NK cells.

[0183] In various embodiments, the method of manufacturing is amenable to scaling to clinically relevant doses.

[0184] In embodiments, the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells act synergistically to kill cancer cells.

[0185] In numerous embodiments, the administering is intravenous, intraarterial, intratumoral, or injected in the vicinity of a tumor.

[0186] In embodiments, the cancer is a blood cancer.

[0187] In some embodiments, the cancer is a solid tumor.

[0188] In various embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.

[0189] Another aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for treating a cancer.

[0190] Yet another aspect of the present disclosure is a method for killing a cancer cell or for inhibiting the proliferation of a cancer cell. The method comprising contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0191] In numerous embodiments, the cancer cell is contacted with the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells simultaneously.

[0192] In embodiments, the cancer cell is contacted with the population of isolated lymphoid lineage cells before being contacted with the population of isolated myeloid lineage cells or the cancer cell is contacted with the population of isolated lymphoid lineage cells after being contacted with the population of isolated myeloid lineage cells.

[0193] In some embodiments, wherein one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR). In some cases, the CAR comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In various cases, the antigen binding region binds to one or more tumor antigens. In these embodiments, the CAR may comprise an antigen binding region that binds to ROR1.

[0194] In various embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine.

[0195] In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, optionally, wherein the cells express a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0196] In embodiments, the isolated lymphoid lineage cells comprise cytotoxic lymphocytes. In some cases, the isolated lymphoid lineage cells comprising cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells. In various cases, the cytotoxic lymphocyte targets and kills cancer cells, e.g., the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. The cytotoxic lymphocyte has reduced cytotoxicity to an NK-resistant cancer cell. In some cases, the cytotoxic lymphocyte is a Natural Killer (NK) cell, e.g., a mature NK cell, or is a cytotoxic T cell. The cytotoxic lymphocyte may be a Natural killer T (NKT) cell. In these embodiments, the NK cell expresses CD16a and / or the NK cell does not express CD3 and / or the NK cell is CD56bright CD16dim / −. In some cases, the NK cell secretes one or more cytokines selected from interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b). In other case, the cytotoxic lymphocyte is a delta-gamma T cell. In these embodiments, the cytotoxic lymphocyte is further engineered to express a chimeric antigen receptor (CAR) and / or is further engineered to express or overexpress a cytokine.

[0197] In some embodiments, the isolated myeloid lineage cells comprise a megakaryocyte, erythrocyte, mast cell, myeloblast, dendritic cell, basophil, neutrophil, eosinophil, monocyte, or macrophage.

[0198] In various embodiments, the isolated myeloid lineage cells express one or more of CD11b, CD13, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., in amounts that are similar to amounts expressed by PBMC-derived cells.

[0199] In numerous embodiments, the isolated myeloid lineage cells have increased expression of CD80 and / or CD206, which is indicative of an activated state.

[0200] In embodiments, the isolated myeloid lineage cell is a macrophage. In some cases, the macrophage expresses one or more of CD11b, CD68, CD80, CD86, CD163, CD206, and SIRPα in amounts that are similar to amounts expressed by PBMC-derived cells and / or secretes one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells. The macrophage may express one or more of CD34, CD44, CD45, CD73, and CD90. In various cases, the macrophages comprise M1 macrophages and / or M2 macrophages. The M1 macrophages and / or M2 macrophages may secrete one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells.

[0201] In some embodiments, the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0202] In various embodiments, the isolated myeloid lineage cell is further engineered to express a chimeric antigen receptor (CAR).

[0203] In numerous embodiments, the isolated myeloid lineage cell is further engineered to express or overexpress a cytokine.

[0204] In embodiments, the isolated lymphoid lineage cells and the isolated myeloid lineage cells are manufactured by a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor. In cases when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated. In these embodiments, the stem cell may be an induced pluripotent stem (iPSC). In some cases, the isolated lymphoid lineage cell and the isolated myeloid lineage cell are derived from the same iPSC. In various cases, the iPSC comprises a genomic modification that expresses a chimeric antigen receptor (CAR) and / or comprises a genomic modification that expresses or over expresses a cytokine. The iPSC may comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, e.g., a biallelic disruption in a B2M gene; in these cases, the iPSC expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0205] In embodiments, the iPSC was reprogrammed from a somatic cell, and the method further comprises contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors. The one or more reprogramming factors may be selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In some cases, the somatic cell is selected from fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0206] In some embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vitro.

[0207] In various embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vivo.

[0208] Yet a further aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for killing a cancer cell or for inhibiting the proliferation of a cancer cell.

[0209] In an aspect, the present disclosure provides a method for manufacturing a plurality of population of cells comprising a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells for treating a cancer, for killing a cancer cell, and / or for inhibiting the proliferation of a cancer cell. The method a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor.

[0210] In numerous embodiments, when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated.

[0211] In embodiments, the stem cell is an induced pluripotent stem (iPSC). In some cases, the isolated lymphoid lineage cell and the isolated myeloid lineage cell are derived from the same iPSC. The iPSC may comprise a genomic modification that expresses a chimeric antigen receptor (CAR) and / or a genomic modification that expresses or over expresses a cytokine. In various cases, the iPSC comprises a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, e.g., a biallelic disruption in a B2M gene. In these cases, the iPSC expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0212] In some embodiments, the iPSC was reprogrammed from a somatic cell, and the method further comprises contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors. The one or more reprogramming factors may be selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In some case, the somatic cell is selected from fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0213] In various embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR), e.g., which comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In some case, the antigen binding region binds to one or more tumor antigens. In these embodiments, the CAR may comprise an antigen binding region that binds to ROR1. In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine.

[0214] In embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene. In some cases, the cells express a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0215] In some embodiments, the isolated lymphoid lineage cells comprise cytotoxic lymphocytes. In some cases, the isolated lymphoid lineage cells comprising cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells. The cytotoxic lymphocyte targets and kills cancer cells. In various cases, the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. The cytotoxic lymphocyte may have reduced cytotoxicity to an NK-resistant cancer cell. The cytotoxic lymphocyte may be a Natural Killer (NK) cell, e.g., a mature NK cell, or is a cytotoxic T cell The cytotoxic lymphocyte may be a Natural killer T (NKT) cell. The the NK cell may express CD16a and / or the NK cell does not express CD3 and / or the NK cell is CD56bright CD16dim / −. In various cases, the NK cell secretes one or more cytokines selected from interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b). The cytotoxic lymphocyte may be a delta-gamma T cell. In some cases, the cytotoxic lymphocyte is further engineered to express a chimeric antigen receptor (CAR) and / or is further engineered to express or overexpress a cytokine.

[0216] In various embodiments, the isolated myeloid lineage cells comprise a megakaryocyte, erythrocyte, mast cell, myeloblast, dendritic cell, basophil, neutrophil, eosinophil, monocyte, or macrophage.

[0217] In numerous embodiments, the isolated myeloid lineage cells express one or more of CD11b, CD13, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., in amounts that are similar to amounts expressed by PBMC-derived cells.

[0218] In embodiments, the isolated myeloid lineage cells have increased expression of CD80 and / or CD206, which is indicative of an activated state.

[0219] In some embodiments, the isolated myeloid lineage cell is a macrophage. In some cases, the macrophage expresses one or more of CD11b, CD68, CD80, CD86, CD163, CD206, and SIRPα in amounts that are similar to amounts expressed by PBMC-derived cells and / or secretes one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells. In various cases, the macrophage expresses one or more of CD34, CD44, CD45, CD73, and CD90. In these embodiments, the method may further comprise a step of differentiating the macrophages into M1 and / or M2 macrophages, e.g., by exposure to MCSF. The method may also further comprise a step of polarizing the M1 macrophages with interferon gamma (IFN-γ) and / or lipopolysaccharide (LPS) and / or treating the M2 macrophages with IL-4. In various cases, the macrophages comprise M1 macrophages and / or M2 macrophages; the M1 macrophages and / or M2 macrophages may secrete one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells.

[0220] In various embodiments, the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0221] In numerous embodiments, the isolated myeloid lineage cell is further engineered to express a chimeric antigen receptor (CAR).

[0222] In embodiments, the isolated myeloid lineage cell is further engineered to express or overexpress a cytokine.

[0223] In some embodiments, the iPSC was contacted with resveratrol before reprogramming.

[0224] In various embodiments, one or more culturing steps comprise a medium which is serum-free culture medium and / or feeder-free culture medium. In some cases, the serum-free culture medium and / or feeder-free culture medium is an mTeSR™ medium and / or the serum-free culture medium and / or feeder-free culture medium is a StemDiff™ NK medium.

[0225] In numerous embodiments, the adherent culturing vessel is a multi-well plate or a cell culturing flask. In embodiments, the method of manufacturing provides at least 1×106 myeloid lineage cells / ml and at least 3×105 lymphoid lineage cells / ml.

[0226] In some embodiments, the method of manufacturing provides both CD14+ (>95% positive) macrophages and CD56bright / CD16dim NK cells.

[0227] In various embodiments, the method of manufacturing is amenable to scaling to clinically relevant doses.

[0228] In embodiments, the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells act synergistically to kill cancer cells.

[0229] In embodiments, the cancer is a blood cancer.

[0230] In some embodiments, the cancer is a solid tumor.

[0231] In various embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.

[0232] In another aspect, the present disclosure provides a plurality population of cells comprising a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells for treating a cancer, for killing a cancer cell, and / or for inhibiting the proliferation of a cancer cell which were manufactured by any herein-disclosed method.Chimeric Antigen Receptor (CAR)-Bearing Cytotoxic Lymphocytes

[0233] In embodiments, the present cytotoxic lymphocytes are engineered with chimeric antigen receptors (CARs), e.g., the present cytotoxic lymphocytes are CAR-NK cells, CAR-T cells, CAR-myeloid cells, or CAR-mesenchymal stromal / stem cells.

[0234] In embodiments, the cytotoxic lymphocyte, optionally NK cell or T cell, is genetically modified to express a recombinant chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In embodiments, the intracellular signaling domain comprises at least one immune receptor tyrosine-based activation motif (ITAM)-containing domain.

[0235] In embodiments, the intracellular signaling domain is from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0236] In embodiments, the transmembrane domain is from one of CD28 or a CD8.

[0237] In embodiments, the antigen binding region binds one antigen. In embodiments, the binding region binds two antigens.

[0238] In embodiments, the extracellular domain comprising an antigen binding region comprises: (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv). In embodiments, the extracellular domain comprising an antigen binding region comprises two of (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv). In embodiments, the extracellular domain comprising an antigen binding region comprises one of each of: (a) a natural ligand or receptor, or fragment thereof, and (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0239] In embodiments, the antigen binding region binds a tumor antigen.

[0240] In embodiments, the antigen binding region comprises one or more of (i) CD94 / NKG2a, which optionally binds HLA-E on a tumor cell; (ii) CD96, which optionally binds CD155 on a tumor cell; (iii) TIGIT, which optionally binds CD155 or CD112 on a tumor cell; (iv) DNAM-1, which optionally binds CD155 or CD112 on a tumor cell; (v) KIR, which optionally binds HLA class I on a tumor cell; (vi) NKG2D, which optionally binds NKG2D-L on a tumor cell; (vii) CD16 (e.g., CD16a or CD16b), which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; (viii) NKp30, which optionally binds B7-H6 on a tumor cell; (ix) NKp44; and (x) NKp46.

[0241] In embodiments, the antigen binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6, as well as any variant thereof.

[0242] In embodiments, the antigen binding region binds an antigen, e.g., a tumor antigen, selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, as well as any variant thereof. In embodiments, an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, as well as any variant thereof can be used as a single-target CAR, dual-target CAR, mAb, or any combination of any of those.

[0243] In embodiments, the antigen binding region binds two antigen, e.g., two tumor antigens, the antigens being: (a) an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2, as well as any variant thereof and (b) an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2, as well as any variant thereof.

[0244] In embodiments, the antigen binding region binds two antigen, the antigens being: (a) an antigen selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvlll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-1, IGF-I I, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), multiple myeloma or lymphoblastic leukemia antigen, such as one selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5, a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any variant thereof and (b) an antigen selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variantI II (EGFRvl ll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-l, IGF-I I, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), multiple myeloma or lymphoblastic leukemia antigen, such as one selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5, a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any variant thereof.

[0245] In embodiments, the extracellular domain of the recombinant CAR comprises the extracellular domain of an NK cell activating receptor or a scFv.

[0246] In embodiments, the NK cell comprises a gene-edit in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1.

[0247] In embodiments, the gene-edit in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1 is caused by contacting the cell with RNA encoding one or more gene-editing proteins. In embodiments, the gene-edit of causes a reduction or elimination of expression and / or activity of IL-6, NKG2A, NKG2D, KIR, TRAC, PD1, and / or HPK1. In embodiments, the gene-edit causes an increase of expression and / or activity of IL-7, CCL17, CCR4, IL-6R, IL-12, IL-15, and / or TRAIL.

[0248] In embodiments, the cytotoxic lymphocyte, e.g., a T cell, NK cell, further comprises one or more recombinant genes capable of encoding a suicide gene product. In embodiments, the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and an apoptotic signaling protein.

[0249] Any cytotoxic lymphocyte disclosed herein (e.g., manufactured by a method disclosed herein, comprising a gene edit (e.g., in B2M), expressing a high affinity CD16a receptor, and / or expressing a fusion protein comprising B2M polypeptide and an HLA polypeptide) can be further genetically engineered to express a CAR.mRNA Cell Engineering Enables Rapid Prototyping of Macrophage Gene-Editing Strategies for Cancer Immunotherapy Applications

[0250] Macrophages' ability to infiltrate solid tumors and engage in both direct killing of cancer cells and recruitment of other immune cells has made them a promising target for development of next-generation cancer immunotherapies. The innate ability of macrophages to ingest foreign genetic material also facilitates their engineering with formulated nucleic acids, including mRNA. The oncoantigen tyrosine-protein kinase transmembrane receptor ROR1 has garnered interest for its minimal expression in healthy adult cells and overexpression in many malignancies, including solid tumors associated with ovarian, lung, and triple-negative breast cancer.

[0251] In this disclosure, e.g., in Example 10, an mRNA-based platform for rapid prototyping of macrophage engineering approaches is described. Here is shown mRNA delivery to peripheral blood mononuclear cell (PBMC) and iPS cell-derived macrophages for gene editing prototyping and functional assessment of encoded proteins. To develop this platform, macrophages were transfected with unmodified or 5′-methoxyuridine (5-moU)-containing mRNA encoding green fluorescent protein (GFP). Both mRNAs resulted in more than 95% of cells displaying GFP within 4 hours. ROR1-targeting CAR with a CD3 zeta activation domain and 4-1BB costimulatory domain was designed.

[0252] Transfecting mRNA encoding the ROR1-CAR yielded 70% CAR-expressing cells, as measured using PE-labelled ROR1. The ROR1 affinity of rabbit and humanized binding domains was analyzed and the humanized binding domain displayed a 2.5-fold increase in affinity as measured by flow cytometry using PE-labelled ROR1. The human receptor domain, but not the rabbit domain, demonstrated activation when bound to ROR1 as assessed by immunofluorescence of CD3 zeta phosphorylation. The mRNA-encoded ROR1-CAR's functionality was assayed by measuring killing of ROR1-expressing SKOV-3 ovarian cancer cells. Both the rabbit and humanized ROR1 domains of the CAR displayed significantly increased cytotoxicity towards SKOV-3 cells when compared with un-transfected macrophages after a 24-hour co-culture at a 5:1 effector-to-target ratio (p<0.01). The ROR1-CAR sequence was inserted into the AAVS1 safe harbor locus of iPSCs under the control of an SFC promotor, isolated5 biallelic-inserted lines, and the resulting cells were differentiated into macrophages. These results demonstrated an mRNA-based platform for rapid prototyping of macrophage engineering approaches. Transfection of macrophages with mRNA encoding a chimeric antigen receptor (CAR) targeting ROR1 resulted in functional expression in vitro, facilitating optimization of the antibody and co-stimulatory domains to improve protein binding affinity and immune activation. This platform thus enables the assessment and validation of novel macrophage gene editing strategies and is being explored for the development of macrophage-engineering therapies for solid tumor applications.

[0253] Indeed, this method permits screening of a library of CAR constructs in vitro to determine which constructs are readily expressed and which are most functional, e.g., in targeting and / or killing cancer cells. Once a CAR construct has been identified as superior, cells ex vivo may be gene-edited to express the construct and the edited cells may be administered into a subject in need and / or the cells of the subject in need may be gene-edited in vivo such that the construct genetically modify cells within the subject.

[0254] An aspect of the present disclosure is a method for screening constructs capable of being expressed in an in vivo cell and for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; and (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell. Another aspect of the present disclosure is method for screening constructs capable of being expressed in an ex vivo cell and for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) culturing the cell capable of expressing the fusion protein which recognizes and / or binds to a cancer cell until a therapeutic amount of the cell is manufactured.

[0255] A further aspect of the present disclosure is a method for screening constructs capable of being expressed in an ex vivo cell and for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) contacting an ex vivo cell with the synthetic mRNA encoding the gene-editing protein and the repair template encoding the fusion protein which was identified in step (3) as having the ability recognize and / or bind to a cancer cell; and (5) culturing the cell of step (4) until a therapeutic amount of the cell is manufactured.

[0256] An additional aspect of the present disclosure is a method for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; and (4) administering to a subject in need the synthetic mRNA encoding the gene-editing protein and the repair template encoding the fusion protein which has the ability recognize and / or bind to a cancer cell.

[0257] In an aspect, the present disclosure provides a method for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) culturing the cell capable of expressing the fusion protein which recognizes and / or binds to a cancer cell until a therapeutic amount of the cell is manufactured; and (5) administering a therapeutically-effective amount of the cells of step (4) to a subject in need. In another aspect, the present disclosure provides a method for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) contacting an ex vivo cell with the synthetic mRNA encoding the gene-editing protein and the repair template encoding the fusion protein which was identified in step (3) as having the ability recognize and / or bind to a cancer cell; (5) culturing the cell of step (4) until a therapeutic amount of the cell is manufactured; and (6) administering a therapeutically-effective amount of the cells of step (4) to a subject in need.

[0258] In embodiments, the fusion protein that recognizes and / or binds to a cancer cell is a chimeric antigen receptor (CAR).

[0259] In embodiments, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region.

[0260] In embodiments, the intracellular signaling domain comprises at least one immunoreceptor tyrosine-based activation motif (ITAM)-containing domain.

[0261] In embodiments, the intracellular signaling domain is from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0262] In embodiments, the transmembrane domain is from one of CD28 or a CD8.

[0263] In embodiments, the antigen binding region binds one antigen.

[0264] In embodiments, the antigen binding region binds two antigens.

[0265] In embodiments, the extracellular domain comprising an antigen binding region comprises: (a) C natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0266] In embodiments, the extracellular domain comprising an antigen binding region comprises two of (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0267] In embodiments, the extracellular domain comprising an antigen binding region comprises one of each of: (a) a natural ligand or receptor, or fragment thereof, and (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0268] In embodiments, the antigen binding region binds a tumor antigen.

[0269] In embodiments, the antigen binding region comprises one or more of: a. CD94 / NKG2a, which optionally binds HLA-E on a tumor cell; b. CD96, which optionally binds CD155 on a tumor cell; c. TIGIT, which optionally binds CD155 or CD112 on a tumor cell; d. DNAM-1, which optionally binds CD155 or CD112 on a tumor cell; e. KIR, which optionally binds HLA class I on a tumor cell; f. NKG2D, which optionally binds NKG2D-L on a tumor cell; g. CD16a, which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; h. NKp30, which optionally binds B7-H6 on a tumor cell; i. NKp44; and j. NKp46.

[0270] In embodiments, the antigen binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6.

[0271] In embodiments, the antigen binding region binds an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2.

[0272] In embodiments, the antigen binding region binds two antigens, the antigens being: a. an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2 and b. an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2.

[0273] In embodiments, the extracellular domain of the recombinant CAR comprises the extracellular domain of an NK cell activating receptor or a scFv.

[0274] In embodiments, the cell type is of the lymphoid cell lineage or the myeloid cell lineage. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell, or an NK cell, e.g., an NK-T cell. In various cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0275] In embodiments, after gene editing, the cell is a CAR-T cell, CAR-NK cell, a CAR-myeloid cell, or a CAR-mesenchymal stromal / stem cell.

[0276] When gene-editing is performed in vivo, transfection may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System, e.g., which relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.FIG. 30 is a cartoon showing methods for rapid prototyping of macrophage gene-editing strategies for cancer immunotherapies.Immune Silenced, Yet Self-Activating, Proliferative, and Anti-Tumoral Cells

[0278] Autologous engineered cell therapies such as autologous chimeric antigen receptor T-cell (CAR-T) therapies have revolutionized the treatment of hematologic cancers, however they are limited by manufacturing time and variability, the requirement for lymphodepletion, and side effects related to cytokine release. Allogeneic cell therapies derived from gene-edited induced pluripotent stem cells (iPSCs) are being developed to address the challenges associated with autologous engineered cell therapies. These “off-the-shelf” cell therapies contain specific edits designed to reduce immune rejection and to confer enhanced therapeutic properties and greater safety. However, efficient, footprint-free, biallelic targeting of defined loci in iPSCs remains technically challenging with current gene-editing approaches.

[0279] Further, while induced pluripotent stem cells (iPSCs) readily differentiate into a wide variety of cell types both in vitro and in vivo, the development of directed differentiation protocols that reliably yield pure populations of functional cells has proved challenging, in particular when differentiating into cell of the lymphoid or myeloid lineage. Generating functional cytotoxic lymphocytes from iPSCs is of particular interest to support the development of off-the-shelf engineered cell therapies for immune-oncology applications.

[0280] What is needed is improved compositions and methods for generating cellular therapies that can be engineered and produced in a practical manner.

[0281] The present disclosure is based, in part, on the discovery that cytotoxic lymphocytes, of the lymphoid cell lineage, e.g., T cells, NK cells, or cells of the myeloid lineage, e.g., macrophages, or mesenchymal stromal / stem cells, or hematopoietic stem cells can be gene-edited and differentiated, using mRNA- and iPS-based methods, to yield therapeutic cells that are immune silenced, yet self-activating, proliferative, and anti-tumoral.

[0282] Cytotoxic lymphocytes, including T cells and NK cells, are being developed as allogeneic, “off-the-shelf”, cell therapies for the treatment of hematological and solid tumors. Allogenic lymphocyte therapies face challenges, however, including limited expansion potential and limited in vivo persistence due to host immune rejection.

[0283] The resulting lymphocytes were characterized for surface markers via flow cytometry and incubated with cancer cells to assess tumor cell engagement and cytotoxicity. Notably, consistently higher yields of lymphocytes were obtained from the B2M-knockout iPSC line relative to a parental, wild-type iPSC line. Both wild-type and B2M-knockout lymphocytes cells killed 75-90% of K562 cells after 24 hours (effector to target (E:T) ratio of 5:1). Interestingly, cytotoxic lymphocytes derived from B2M-knockout iPSCs exhibited greater K562 cell killing with the addition of IL15 and IL2, while killing by wild-type cells was not controlled by these activating cytokines. Cancer cell killing activity was maintained through cryopreservation, albeit at a reduced level (15-40% reduction in activity). Accordingly, B2M-knockout iPSCs of the present disclosure are an ideal source of cytotoxic lymphocytes for the development of “off-the-shelf” allogeneic cell therapies for the treatment of cancer and without substantial host immune rejection.

[0284] In one aspect, there is provided cell comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a loss of function, optionally in both alleles, of the B2M gene, wherein the cell is a cytotoxic lymphocyte from a lymphoid lineage cell or the cell is a myeloid lineage cell. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage. In embodiments the cytotoxic lymphocyte is a NK cell.

[0285] The present cytotoxic lymphocyte is sometimes referred to herein as an “engineered cytotoxic lymphocyte”.

[0286] An aspect of the present disclosure is a method for manufacturing a population of cells that is enriched for cytotoxic lymphocytes. The method comprises steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes. In this aspect, steps (5) and (6) occur in an adherent culturing vessel. When CD34+ cells are selected, the embryoid bodies may be first chemically and / or mechanically dissociated. In embodiments, the stem cell is an induced pluripotent stem (iPSC). In this aspect, the stem cell has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene. In some cases, the stem cell has a biallelic disruption in a B2M gene.

[0287] In another aspect, there is provided a method of making an engineered cell comprising a disrupted B2M gene, the method, comprising (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins; and (c) differentiating the iPS cell into a cytotoxic lymphocyte, e.g., a cell of the lymphoid cell lineage or into a cell of the myeloid cell lineage. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0288] An aspect of the present disclosure is a method for killing a cancer cell. The method comprising steps of: (1) obtaining a herein-disclosed cytotoxic lymphocyte which was derived from a stem cell has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a biallelic B2M knockout, and (2) contacting cytotoxic lymphocyte with the cancer. In some cases, the cancer cell is in vivo.

[0289] Yet another aspect of the present disclosure is a method for treating a cancer patient in need thereof. The method comprising a step of administering to the cancer patient a therapeutically-effective amounts of a herein-disclosed cytotoxic lymphocyte which was derived from a stem cell has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a biallelic B2M knockout.

[0290] In some cases, mRNA-reprogrammed iPSC lines with a biallelic knockouts of the beta-2 microglobulin (B2M) gene, a key component of MHC class I molecules, are obtained using an mRNA-encoded chromatin context-sensitive gene-editing endonuclease. The B2M-knockout iPSCs may be differentiated using a novel, fully suspension process that replaces specialized micropatterned culture vessels with a spheroid culture step. Additional details regarding B2M knockout iPSCs useful in the present disclosure are described in PCT / US2022 / 019020, the contents of which are incorporated herein by reference in its entirety.

[0291] In another aspect, there is provided a method of treating cancer, comprising (a) obtaining an isolated cytotoxic lymphocyte comprising a genetically engineered disruption in a B2M gene; and (b) administering the isolated cytotoxic lymphocyte to a subject in need thereof, wherein the cytotoxic lymphocyte is selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.Immune Silencing

[0292] In embodiments, the present cytotoxic lymphocyte is engineered to evade recognition and / or clearance by a host immune system. In embodiments, the present cytotoxic lymphocyte is a stealth cytotoxic lymphocyte. In embodiments, the present cytotoxic lymphocyte is not substantially recognized by an immune system upon administration to a subject.

[0293] In embodiments, the present cytotoxic lymphocyte has reduced or eliminated susceptibility to cell killing by T cells as compared to a cytotoxic lymphocyte which does not comprise a genetically engineered disruption in the B2M gene. In embodiments, the present cytotoxic lymphocyte has reduced or eliminated susceptibility to cell killing by other cytotoxic lymphocytes as compared to another cytotoxic lymphocyte which comprises a genetically engineered disruption in the B2M gene. In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of B2M is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of B2M is reduced or inhibited.

[0294] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of MHC class I is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of MHC class I is reduced or inhibited.

[0295] In embodiments, the B2M gene is a human B2M gene (e.g., NCBI Reference Sequence: NG_012920). The sequence of the B2M gene of various embodiments is provided in the EXAMPLES section herein. B2M, is the light chain of MHC class I molecules, and as such an integral part of the major histocompatibility complex. In humans, B2M is encoded by the b2m gene which is located on chromosome 15. The human protein is composed of 119 amino acids and has a molecular weight of 11.8 kilodaltons (e.g., UniProtKB—P61769). The amino acid sequence of human beta-2-microglobulin (B2M) is:(SEQ ID NO: 13)MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.

[0296] In embodiments, the present cytotoxic lymphocyte has genetically engineered disruptions of all substantially all copies of the B2M gene. In embodiments, the present cytotoxic lymphocyte has a loss of function of the B2M gene. In embodiments, the present cytotoxic lymphocyte has a loss of function of both alleles of the B2M gene.

[0297] In embodiments, the genetically engineered disruption of the B2M gene is in exon 3 of human B2M. In embodiments, the genetically engineered disruption of the B2M gene is a deletion. In embodiments, the deletion is about 10 to about 20 nucleotides. In embodiments, the deletion is near nucleotides 500 to 550 of the human B2M gene. In embodiments, the deletion is of the sequence TTGACTTACTGAAG (SEQ ID NO: 14), or a functional equivalent thereof.

[0298] In embodiments, the present cytotoxic lymphocyte has downregulated MHC class I expression and / or activity.

[0299] In embodiments, the genetically engineered disruption of B2M comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins.

[0300] In embodiments, the present cytotoxic lymphocyte is engineered to be further immune silenced, e.g., in addition to B2M (MHC Class I) disruption. In embodiments, the present cytotoxic lymphocyte is engineered to be disrupted at the human MHC II transactivator (CIITA) gene (NCBI Reference Sequence: NG_009628.1).

[0301] In embodiments, the present cytotoxic lymphocyte has downregulated MHC class II expression and / or activity.

[0302] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of CIITA is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of CIITA is reduced or inhibited.

[0303] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of MHC class II is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of MHC class II is reduced or inhibited.

[0304] In embodiments, the genetically engineered disruption of CIITA comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins.

[0305] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of B2M and CIITA is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of B2M and CIITA is reduced or inhibited.

[0306] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of MHC class I and MHC class II are reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of MHC class I and MHC class II are reduced or inhibited.

[0307] In embodiments, the genetically engineered disruption of B2M and CIITA comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins. In embodiments, the present cytotoxic lymphocyte comprises a genetically engineered alteration in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0308] In embodiments, the cytotoxic lymphocyte expresses a fusion protein comprising a B2M polypeptide and a HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide. The fusion protein may be expressed by insertion of a repair template into a single or double strand break of the B2M gene; in some cases, the repair template comprises the coding sequence for B2M and the HLA gene. Notably, the fusion protein replaces endogenous B2M and HLA pairs expressed by a cytotoxic lymphocyte, thereby reducing the likelihood that the cytotoxic lymphocyte will be reduced or eliminated by a host cytotoxic lymphocyte.

[0309] In embodiments, the present cytotoxic lymphocyte does not comprise a genetically engineered alteration in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0310] In embodiments, the genetically engineered alteration is a genetically engineered reduction or elimination in expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0311] In embodiments, the genetically engineered alteration is a genetically engineered increase in expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0312] In embodiments, the genetically engineered disruption of B2M is combined with a genetically engineered expression of a fusion between B2M or a fragment thereof and one or more genes and / or fragments thereof selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0313] In embodiments, the B2M or fragment thereof and one or more genes and / or fragments thereof are separated by a linker region. In embodiments the linker is (G4S)3.

[0314] In embodiments, the genetically engineered alteration is a genetically engineered increase in expression and / or activity of one or more genes selected from IL-2, IL-15, IL-21. In embodiments, the IL-15 contains the N72D mutation. In embodiments, the IL-15 is fused to the cytokine binding domain of IL-15Rα.

[0315] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of negative regulators of IL-15 signaling are reduced or inhibited. In embodiments, the negative regulator of IL-15 signaling is the CISH protein. In embodiments, the reduction or inhibition of negative regulators of IL-15 signaling is achieved by genetically engineered disruption of the CISH gene. The Cytokine-inducible SH2-containing protein (CISH) gene is found at gene ID: NG_023194.1.

[0316] In embodiments, the genetically engineered disruption of CISH comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins.Methods of Making B2M Knockout Cells

[0317] In aspects, the present disclosure provides a method of making an engineered cell comprising a disrupted B2M gene, the method comprising: (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a beta-2-microglobulin (B2M) gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins; and (c) differentiating the iPS cell into a cytotoxic lymphocyte, e.g., a cell of the lymphoid cell lineage, or into a cell of the myeloid cell lineage, or a mesenchymal stem / stromal cell, or a hematopoietic stem cell. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0318] In embodiments, the method further comprises disrupting a CIITA gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins.

[0319] In embodiments, the cytotoxic lymphocyte is an NK cell.

[0320] In embodiments, the somatic cell is a fibroblast or keratinocyte.

[0321] In embodiments, the method provides an increased proliferation rate of iPS cells as compared to the rate of iPS cells without a disruption of the B2M gene.

[0322] In embodiments, the method provides an increased proliferation rate of differentiating cells along a lymphoid lineage cell as compared to the rate of iPS cells without a disruption of the B2M gene.

[0323] In embodiments, the method provides an increased expansion of differentiating cells along a lymphoid lineage cell as compared to the rate of iPS cells without a disruption of the B2M gene.

[0324] In embodiments, the differentiating comprises embryoid body-based hematopoietic commitment. In embodiments, the differentiating comprises enrichment of CD34+ cells. In embodiments, the differentiating comprises differentiating into CD5+ / CD7+ common lymphoid progenitors.

[0325] In embodiments, the method yields CD56dim CD16+ NK cells.

[0326] In embodiments, the RNA is associated with one or more lipid selected from and / or Formulae I-XVI.Differentiation of iPSCs into Monocytes and Macrophages

[0327] In one aspect, the present disclosure relates to induced pluripotent stem cell (iPSC)-derived monocytes that can be differentiated into functional M1 and M2 macrophages with enhanced cytokine secretion and tumor cell-killing activity.

[0328] Although cancer immunotherapy has advanced rapidly over the past two decades, with several autologous chimeric antigen receptor (CAR)-T cell therapies approved for the treatment of hematologic cancers, CAR-T cells have shown limited activity against solid tumors, in part due to the immunosuppressive nature of the tumor microenvironment preventing CAR-T cell infiltration. This has led to investigation of other immune cells as alternatives to T-cell-based therapies, including monocytes and monocyte-derived macrophages, which exhibit innate tumor-infiltration properties. Disclosed herein is a new and useful process for differentiating pluripotent stem cells along a myeloid lineage, and generated populations of cells with characteristics of monocytes and M1 and M2 macrophages, including cytokine secretion and tumor cell-killing activity. mRNA-reprogrammed human induced pluripotent stem cells (iPSCs) were differentiated into monocytes using a 28-day monolayer protocol. Beginning on day 14, cells were harvested every 3-4 days. CD14+ isolation yielded >95% CD14+ cells with an average yield of 4.1×104 cells per cm2 per harvest. iPSC-derived monocytes were compared to peripheral blood mononuclear cell (PBMC)-derived monocytes for expression of key hematopoietic and myeloid-lineage markers CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα. iPSC-derived monocytes showed similar expression of CD11b, CD14, CD33, CD45, and CD163 compared to PBMC-derived monocytes, and increased expression of markers indicative of an activated state: CD80 and CD206. Compared to PBMC-derived monocytes, iPSC-derived monocytes showed both higher viability in culture and superior recovery from cryopreservation. iPSC-derived monocytes were further differentiated into macrophages by exposure to MCSF for 3-4 days, and were assessed for their ability to polarize, secrete pro- and anti-inflammatory cytokines, and for cytotoxic activity when co-cultured with cancer cells. M1 macrophages were polarized with interferon gamma (IFN-γ, 50 ng / mL) and lipopolysaccharide (LPS, 10 ng / mL) for 48 hours, while M2 macrophages were treated with IL-4 (10 ng / mL) for 48 hours. iPSC-derived monocytes differentiated into macrophages with >90% efficacy, as assessed by cell adherence, morphology, and surface marker expression (CD14, CD45, CD163). M1 and M2 polarized iPSC-derived macrophages secreted similar levels of TNFα, IL-12p70, and IL-10 compared to PBMC-derived macrophages. iPSC-derived macrophages killed 45% of U2OS cancer cells in vitro after 24 hours at an E:T ratio of 5:1. Disclosed herein is a process for differentiating mRNA-reprogrammed iPSCs into cytotoxic macrophages. The mRNA reprogramming and differentiation processes are virus-free and DNA-free, avoiding any potential risk of vector integration. This disclosure provides proof of concept that mRNA-reprogrammed iPSCs represent a viable source of macrophages for the development of therapies to treat various indications, including solid tumors. The process is illustrated in FIG. 42.

[0329] Monocytes are a type of leukocyte, or white blood cell. They are the largest type of leukocyte and can differentiate into macrophages and conventional dendritic cells. As a part of the vertebrate innate immune system monocytes also influence adaptive immune responses and exert tissue repair functions. There are at least three subclasses of monocytes in human blood based on their phenotypic receptors: The classical monocyte is characterized by high level expression of the CD14 cell surface receptor (CD14++ CD16− monocyte), the non-classical monocyte shows low level expression of CD14 and additional co-expression of the CD16 receptor (CD14+CD16++ monocyte), and the intermediate monocyte expresses high levels of CD14 and low levels of CD16 (CD14++CD16+ monocytes).

[0330] Monocytes are mechanically active cells and migrate from blood to an inflammatory site to perform their functions. In general, monocytes and their macrophage and dendritic cell progeny serve three main functions in the immune system: these are phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of microbes and particles followed by digestion and destruction of this material. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter.

[0331] Macrophages engulf and digest pathogens, such as cancer cells, microbes, cellular debris, and foreign substances, which do not have proteins that are specific to healthy body cells on their surface, via phagocytosis.

[0332] Macrophage that encourages inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages. M1 macrophages are classically activated, typically by IFN-γ or lipopolysaccharide (LPS), and produce proinflammatory cytokines, phagocytize microbes, and initiate an immune response. M1 macrophages produce nitric oxide (NO) or reactive oxygen intermediates (ROI) to protect against bacteria and viruses. M2 macrophages are alternatively activated by exposure to certain cytokines such as IL-4, IL-10, or IL-13. M2 macrophages will produce either polyamines to induce proliferation or proline to induce collagen production. These macrophages are associated with wound healing and tissue repair. iPSCs obtained by a herein-disclosed method may be differentiated into a monocyte which can be further differentiated into a macrophage, e.g., an M1 or M2 macrophage.

[0333] The general steps for this process comprise (1) iPSC to monocyte differentiation, (2) CD14+ magnetic bead positive selection, and (3) monocyte to macrophage differentiation. These general steps are, respectively, shown in FIG. 43A, FIG. 43B, and FIG. 43C.

[0334] As shown in FIG. 43A, embryonic stem cells (ES cells) may be used as starting material for the process rather than iPSCs.

[0335] Beginning on day 14, monocyte cells can be harvested and continue to be harvested every 3-4 days. Additional details regarding differentiation of iPSCs to monocytes is found at the World Wide Web (www) stemcell.com / stemdiff-monocyte-kit.html, the contents of which are incorporated herein by reference in its entirety.

[0336] As shown in FIG. 43B, CD14+ cells, which comprise monocytes and macrophages, are separated using suitable reagents.

[0337] Finally, as shown in FIG. 43C, monocytes are cultured under conditions such that they differentiate into macrophages, e.g., by activation with macrophage colony-stimulating factor (M-CSF). Additional details regarding differentiation of monocytes to macrophage is found at the World Wide Web (www) stemcell.com / immunocult-sf-macrophage-medium.html, the contents of which are incorporated herein by reference in its entirety.

[0338] M1 macrophages can be then polarized into M1 macrophages with interferon gamma (IFN-γ, 50 ng / mL) and lipopolysaccharide (LPS, 10 ng / mL) for 48 hours, whereas macrophages are treated with IL-4 (10 ng / mL) for 48 hours to generate M2 macrophages.

[0339] Mature differentiated cells can be reprogrammed and dedifferentiated into embryonic-like cells, with embryonic stem cell-like properties. Fibroblast cells can be reversed into pluripotency via, for example, retroviral transduction of certain transcription factors or transfection of synthetic nucleic acids encoding transcription factors, resulting in iPSCs. In some embodiments, iPSCs are generated from various tissues, including fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells. In some embodiments, iPSCs are generated via transfection of synthetic nucleic acids encoding the transcription factors Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein and biologically active fragments, analogues, variants and family-members thereof. In some embodiments, iPSCs are generated via transfection of synthetic nucleic acids encoding miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0340] The generation of iPSCs depends on the transduction of specific transcription factors into the somatic cell genome via vectors for its reprogramming.

[0341] In various cases, transfection of a cell with synthetic nucleic acids for reprogramming may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In some embodiments, following iPSC generation, cells are assessed via pluripotency assays, including morphological and histological analysis, and certain gene expression profiles, proving their ability to differentiate into tissues derived from the three germ layers and teratoma formation. In some embodiments, teratoma assays involve injection of iPSCs into immunocompromised experimental animals and subsequent formed tissue analysis to assess teratoma formation.

[0343] In embodiments, the iPSC is derived from a human. In embodiments, the iPSC is derived from a subject who is not intended to receive the therapy. In embodiments, the iPSC is allogeneic to a patient intended to receive the therapy. In embodiments, the iPSC is from a master cell bank.

[0344] Stem cells have the ability to self-renew and differentiate into multiple cell types and so have applications in regenerative medicine.

[0345] In embodiments, the iPSCs, the monocytes, and / or the macrophage is further gene-edited, as disclosed herein. In some embodiments, the iPSC was gene-edited contemporaneously with being reprogrammed, e.g., from a fibroblast. In various embodiments, the iPSC was gene-edited before being reprogrammed, e.g., from a fibroblast. In other embodiments, the iPSC was gene-edited after being reprogrammed, e.g., from a fibroblast.

[0346] In some aspects of the present disclosure, a macrophage that is administered to a patient, e.g., for killing cancer cell, was ultimately derived from the patient. That is to say, a keratinocyte (as an example) is obtained from the patient and this keratinocyte is reprogrammed into an IPSC which is differentiated into a monocyte and further differentiated into an M1 or M2 macrophage. By starting with the patient's own cells, the macrophage administered back to the patient could be considered to be autologous.

[0347] The use of autologous macrophages in therapeutic applications is safe because the cells will not elicit an immune response. However, it may be difficult to obtain a large amount of bone marrow or adipose tissue from the subject. Autologous macrophages may also have reduced therapeutic efficacy resulting in poor clinical outcomes. Additionally, if macrophages are needed urgently, there may not be time to extract and expand autologous macrophages from a subject.

[0348] Thus, the use of allogeneic macrophages is an attractive alternative because donors can be prescreened for having cells with a high therapeutic potential. Allogeneic macrophages can be prepared on a clinical scale, assayed for therapeutic potential after production and stored in usable clinical doses that can be used readily for urgent therapeutic applications. In some embodiments, the present macrophages are allogeneic.

[0349] Thus, obtaining macrophages by reprogramming iPSCs, which may be obtained by reprogramming human somatic cells, is an attractive alternative. In some embodiments, iPSCs are obtained monocytes are generated via cell reprogramming with non-immunogenic messenger RNA (mRNA) encoding one or more reprogramming factors in a defined, animal-component-free process. The monocytes are further differentiated into macrophages. See, e.g., FIG. 42.

[0350] In some embodiments, monocytes and / or macrophages are checked for safety using one or more of bacterial and fungal tests, Mycoplasma test, adventitious viral agent test, and tumorigenicity assay (karyotype analysis, teratoma formation assay, soft agar assay, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR)).

[0351] In embodiments, the monocyte or macrophage has been altered to reduce expression of MHC molecules. In embodiments, the alteration is enabled by gene editing. In embodiments, the MHC molecules are MHC class I molecules. In embodiments, the expression of MHC class I molecules is reduced by gene editing the B2M gene. In some cases, the gene editing occurs in an iPSC cell that is a progenitor of the monocyte or macrophage.The Immunosuppressive TTAGGG Motif Improves Homology-Directed Insertion of DNA Sequences in Human Primary and Induced Pluripotent Stem (iPS) Cells

[0352] In another aspect, the present disclosure relates to use of TTAGGG motif for decreasing synthetic oligodeoxynucleotides (ODNs)-related activation of pro-inflammatory responses; with decreasing the pro-inflammatory responses leading to higher transgene insertion efficiency.

[0353] Double stranded synthetic oligodeoxynucleotides (dsODNs) have been used as repair templates in gene-editing applications to insert transgenic sequences into defined genomic loci, albeit with low efficiency. Cells engineered in this way are of interest for many therapeutic applications, including allogeneic NK and T cells engineered to express stealthing proteins, cytokines, and chimeric antigen receptors (CARs) for the treatment of a variety of cancers. To increase the efficiency of integration, gene-editing proteins can be co-expressed to create a double-strand break at the target locus. However, recognition of dsODNs by pattern recognition receptors activates signaling cascades resulting in the production of cytokines, including type I interferons such as IFIT1-3 and IFN-β. This immune response can lead to cell cycle arrest, differentiation, and apoptosis and may contribute to low insertion efficiency observed in primary and iPS cells. It has been shown in human immune cells that co-delivery of a short ODN comprising the immunosuppressive motif, TTAGGG, which is found in mammalian telomeric DNA, inhibits the activation of the damage-associate molecular pattern (DAMP) pathway in response to cytosolic DNA. This ODN competitively binds to inflammasomes and reduces the secretion of proinflammatory cytokines. The present disclosure relates to dsODNs comprising the TTAGGG motif for decreasing dsODN-related activation of a pro-inflammatory response in human cells, with decreasing the pro-inflammatory responses leading to higher transgene insertion efficiency. As disclosed herein, the TTAGGG motif is incorporated either at the 5′ end of dsODNs or delivered separately on a short single-stranded ODN (A151). Human primary fibroblasts, iMSCs and iPSCs were electroporated with a dsODN encoding a GFP reporter and containing an SfoI restriction site. Upregulation of pro-inflammatory markers including IFIT1-3, was measured by RT-PCR. A 29-fold higher expression of IFIT1 and IFIT3 was observed in cells electroporated with dsODNs than in untreated controls. Interestingly, including TTAGGG motifs at the 5′-ends of the dsODNs limited the upregulation of IFIT1 and IFIT3 to 10- and 15-fold, respectively, while co-delivery of the TTAGGG motif prevented their upregulation altogether. A gene-editing endonuclease targeting the AAVS1 safe-harbor locus on chromosome 19 was then used to investigate the impact of the TTAGGG motif on the insertion of transgenes at this site. The TTAGGG motif (whether incorporated in the dsODN or co-transfected in the form of the A151 ODN) resulted in approximately 50% higher viability and approximately 50% more GFP-positive cells than when the motif was not present. This disclosure provides proof of concept that the herein-disclosed immunosuppressive sequences increases ODN insertion efficiency and improves cell viability and is therefore a powerful tool for therapeutic knock-in applications, including the generation of knock-in iPS cell lines.

[0354] The process of including a TTAGGG motif in an dsODN for reducing an immune response is illustrated in FIG. 54. As shown in FIG. 54, the TTAGGG motif, found in telomeric DNA, is incorporated onto the 5′ end of a double stranded repair template in primary, iMS and iPS cells. The motif is recognized by, and competitively binds to Pattern Recognition receptors in the cytoplasm of the cells to lessen the immune response mounted against the double stranded repair templates.

[0355] The TTAGGG motif may be provided at the 5′ end of a dsODN that serves as a repair template. The TTAGGG motif may be provided at the 3′ end of a dsODN that serves as a repair template. The TTAGGG motif may be provided at both the 3′ end and the 5′ end of a dsODN that serves as a repair template. The dsODN that serves as a repair template may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the TTAGGG motif.

[0356] As disclosed-herein, the TTAGGG motif may be provided to a cell separately from the dsODN on a short single-stranded synthetic oligodeoxynucleotides (e.g., A151). The A151 ssODN comprise four repeats of the TTAGGG motif and the sequence of TTAGGGTTAGGGTTAGGGTTAGGG (SEQ ID NO: 15). In other cases, a single-stranded synthetic oligodeoxynucleotide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the TTAGGG motif. In some case, the TTAGGG motif may be provided to a cell separately from the dsODN on a short double-stranded synthetic oligodeoxynucleotides. In embodiments, the double-stranded synthetic oligodeoxynucleotide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the TTAGGG motif.

[0357] In some embodiments, the cells that are transfected with a TTAGG-containing ODN (either as a repair template or as separate ODN) are skin cells, pluripotent stem cells, embryonic stem cells, iPSCs, MSCs (including iMSCs), mesenchymal stromal / stem cells, hematopoietic cells, hematopoietic stem cells, lymphocytes, β-cells, T-cells (including CAR-T), NK cell (including CAR-NK), monocytes, macrophages (including CAR-myeloid cells and CAR-mesenchymal stromal / stem cells), retinal pigmented epithelial cells, hematopoietic cells, a hematopoietic stem cells, myeloid cells, tumor-infiltrating lymphocytes, marrow-infiltrating lymphocytes, a peripheral blood lymphocytes, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells.

[0358] In embodiments, gene-editing a cell comprises contacting the cell with synthetic nucleic acid encoding one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyz (SEQ ID NO: 16), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.

[0359] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17) or LTPvQVVAIAwxyzGTHG (SEQ ID NO: 18) and is from 36 to 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20) and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In some embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C.

[0360] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 22) and is from 36 to 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), α is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, α is selected from GHGG (SEQ ID NO: 31), HGSG (SEQ ID NO: 32), HGGG (SEQ ID NO: 33), GGHD (SEQ ID NO: 34), GAHD (SEQ ID NO: 35), AHDG (SEQ ID NO: 36), PHDG (SEQ ID NO: 37), GPHD (SEQ ID NO: 38), GHGP (SEQ ID NO: 39), PHGG (SEQ ID NO: 40), PHGP (SEQ ID NO: 41), AHGA (SEQ ID NO: 42), LHGA (SEQ ID NO: 43), VHGA (SEQ ID NO: 44), IVHG (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), RHGD (SEQ ID NO: 47), RDHG (SEQ ID NO: 48), RHGE (SEQ ID NO: 49), HRGE (SEQ ID NO: 50), RHGD (SEQ ID NO: 47), HRGD (SEQ ID NO: 51), GPYE (SEQ ID NO: 52), NHGG (SEQ ID NO: 53), THGG (SEQ ID NO: 54), GTHG (SEQ ID NO: 21), GSGS (SEQ ID NO: 56), GSGG (SEQ ID NO: 57), GGGG (SEQ ID NO: 58), GRGG (SEQ ID NO: 59), and GKGG (SEQ ID NO: 60). In some embodiments, a gene-editing protein comprises a C-terminal GTHG produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C.

[0361] In some cases, a cell is contacted with a demethylating agent during the process of gene-editing. In embodiments, the demethylating agent is selected from 5-azacitidine and 5-aza-2′-deoxycitidine (decitabine).

[0362] In some embodiments, the gene-editing protein comprises: (a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13; and (b) the nuclease domain comprising a catalytic domain, the catalytic domain comprising a hybrid of the catalytic domains of Fokl and Stsl, comprising the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Fokl with at least one of the domains of Fokl being substituted in whole or in part with the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Stsl and optionally comprising at least one mutation.

[0363] In various embodiments, the cell is transfected (e.g., contacted) with a synthetic nucleic acid encoding the gene-editing protein at about 30° C. to about 35° C., e.g., without limitation about 33° C. In embodiments, the contacting occurs at about 30° C. In some embodiments, the contacting occurs at about 31° C. In some embodiments, the contacting occurs at about 32° C. In some embodiments, the contacting occurs at about 33° C. In some embodiments, the contacting occurs at about 34° C. In some embodiments, the contacting occurs at about 35° C. In embodiments, the gene-editing protein is functionally temperature-switchable. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. to about 35° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 31° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 32° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 33° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 34° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 35° C.

[0364] In embodiments, the gene-edited cell, as disclosed herein, is also reprogrammed, as disclosed herein. In some embodiments, the gene-editing is contemporaneously with reprogramming. In various embodiments, the gene-editing is before the reprogramming. In other embodiments, the gene-editing is after the reprogramming. In cases when a cell is reprogramming, the cell may be a differentiated or a non-pluripotent cell. In some cases, the differentiated or a non-pluripotent cell is a skin cell (e.g., a fibroblast or a keratinocyte).Resveratrol

[0365] In yet another aspect, the present disclosure relates to use of Resveratrol treatment prior to transfection, e.g., with a synthetic nucleic acid encoding a gene-editing protein, and / or after transfection, e.g., with a synthetic nucleic acid encoding a gene-editing protein.

[0366] Gene editing technology, which enables the precision modification of DNA in living cells, is being developed for the treatment of various diseases, including genetic diseases and cancer. Gene editing commonly employs sequence-specific endonucleases to create double strand breaks in genomic DNA and relies on the cell's DNA repair mechanisms to apply the desired changes. Precise sequence modifications, such as single-base changes, rely on the homology directed repair (HDR) mechanism. Despite its essential role in gene repair, HDR occurs at a very low frequency in many cells compared to other repair mechanisms. The present disclosure discloses the impact of resveratrol, a small molecule extracted from grape skin, that promotes the expression of key HDR factors and induces cell cycle arrest at S phase in porcine fetal fibroblasts, on single-base editing efficiency in primary human fibroblasts. Following treatment with resveratrol, fibroblasts was co-transfected with mRNA encoding a chromatin context-sensitive gene-editing protein targeting the AAVS1 safe-harbor locus, and a single-stranded DNA repair template designed to introduce a SfoI restriction-enzyme site through a G-to-C mutation. Single-base editing efficiency was determined by restriction fragment length polymorphism (RFLP) analysis. Resveratrol treatment prior to transfection increased the S and G2-phase population 2.3-fold and increased HDR efficiency 2-fold compared to untreated cells. Application of resveratrol after transfection (i.e., no cell cycle synchronization) yielded further improvement in single-base editing efficiency (>2-fold), suggesting that the effects of resveratrol on HDR are not confined to cell-cycle control. This disclosure provides proof of concept that Resveratrol treatment provides a straightforward method for improving HDR efficiency in primary human fibroblasts and serves as a useful tool in the development of HDR-based gene-editing therapies.

[0367] The process of contacting Resveratrol with a cell in advance of gene editing is illustrated in FIG. 58. As shown in FIG. 58, Resveratrol arrests the cell in S or G2 phase and enhances the efficiency of subsequent gene-editing.

[0368] Notably, Resveratrol treatment before transfecting with a gene-editing machinery (e.g., a synthetic nucleic acid encoding a gene-editing protein) appears to arrest the majority of cells in S / G2; this pre-treatment enhances the efficiency of subsequent gene-editing. Also, Resveratrol treatment after transfecting with a gene-editing machinery (e.g., a synthetic nucleic acid encoding a gene-editing protein) enhances the efficiency of ongoing gene-editing. Therefore, in embodiments, cells may be pre-treated with Resveratrol and / or post-treated with Resveratrol.

[0369] Any of the herein-disclosed gene-editing methods may comprise pre-treatment with Resveratrol and / or post-treated with Resveratrol.Reprogramming Methods

[0370] In embodiments, the present disclosure relates to RNA-based modifications, e.g., reprogramming and / or gene-editing. In some embodiments, an RNA molecule encodes a gene-editing protein. In some embodiments, an RNA molecule encodes a reprogramming factor.

[0371] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides.

[0372] In various embodiments, the present invention relates to the reprogramming of iPSCs to Monocytes, which can then be further differentiated into M1 and / or M2 macrophages, using non-viral, RNA-based means. iPSCs, namely pluripotent or less differentiated cells, can be reprogrammed from non-pluripotent or differentiated cells, including fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0373] In some embodiments, the method for reprogramming a non-pluripotent cell comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and wherein step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0374] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed to a less differentiated state; and wherein step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0375] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors; and wherein step (c) is performed at least twice and the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections to result in the cell being reprogrammed to a less differentiated state and occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0376] In some embodiments, the method for reprogramming a non-pluripotent cell, comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and wherein step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell.

[0377] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed to a less differentiated state; and wherein step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell to a less differentiated state.

[0378] In some embodiments, the method for reprogramming a non-pluripotent cell, comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; (c) transfecting the non-pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (d) repeating step (c) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the non-pluripotent cell being reprogrammed, wherein steps (c) and (d) occur in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

[0379] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (d) repeating step (c) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state, wherein steps (c) and (d) occur in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0380] In some embodiments, the method for reprogramming a non-pluripotent cell comprises: (a) providing a non-pluripotent cell, the non-pluripotent cell being derived from a biopsy of a human subject; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with a synthetic RNA molecule, wherein: the synthetic RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the non-pluripotent cell expressing the one or more reprogramming factor(s) which reprograms the non-pluripotent cell; and step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

[0381] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) providing a non-pluripotent cell; (b) culturing the cell; and (c) transfecting the cell with a synthetic RNA molecule, wherein: the RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state, and step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell to a less differentiated state.

[0382] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) providing a non-pluripotent cell; (b) culturing the cell in a medium containing ingredients that support reprogramming of the cell to a less differentiated state; and (c) transfecting the cell with a synthetic RNA molecule, wherein: the RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state, and step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a feeder cell conditioned medium.

[0383] In some embodiments, the method for reprogramming a cell to a less differentiated state comprises: (a) providing a non-pluripotent cell; (b) culturing the cell in a medium containing albumin and ingredients that support reprogramming of the cell to a less differentiated state, wherein the albumin is treated with an ion-exchange resin or charcoal; (c) transfecting the cell with a synthetic RNA molecule, wherein the RNA molecule encoding one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, wherein the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state.

[0384] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) culturing a differentiated cell with a reprogramming medium; (b) transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (c) repeating step (b) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state, wherein steps (a)-(c) are performed without using feeder cells and occur in the presence of a feeder cell conditioned medium.

[0385] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin is treated with an ion-exchange resin or charcoal; b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and c. repeating step (b) at least twice during 5 consecutive days to result in the cell being reprogrammed to a less differentiated state.

[0386] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin is treated with sodium octanoate; brought to a temperature of at least about 40° C.; and treated with an ion-exchange resin or charcoal; b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding one or more reprogramming transcription factors and wherein the transfecting results in the cell expressing the one or more synthetic RNA molecules; and c. repeating step (b) at least twice during about 5 consecutive days to result in the cell being reprogrammed to a less differentiated state. In embodiments, the reprogramming is non-viral. In embodiments, the reprogramming factor is one or more of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0387] In some embodiments, iPSCs are obtained and Monocytes, which can then be further differentiated into M1 and / or M2 macrophages, are generated via cell reprogramming with non-immunogenic messenger RNA (mRNA) encoding one or more reprogramming factors in a defined, animal component-free process. In some embodiments the process is immunosuppressant-free. In some embodiments, the process is animal component-free. In some embodiments, the process is defined. In some embodiments, iPSCs are generated from adult human dermal fibroblasts using a high-efficiency, immunosuppressant-free mRNA-based protocol, whereupon iPSCs are differentiated into monocytes using a 28-day monolayer protocol. Beginning on day 14, cells can be harvested every 3-4 days. CD14+ isolation yielded >95% CD14+ cells with an average yield of 4.1×104 cells per cm2 per harvest. iPSC-derived monocytes were compared to peripheral blood mononuclear cell (PBMC)-derived monocytes for expression of key hematopoietic and myeloid-lineage markers CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα. In some embodiments, the differentiated monocytes are characterized by downregulation of Nanog and Oct4 and / or changes in expression of CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., relative to the source cells. In some embodiments, rtPCR analysis is used to characterize monocytes. In some embodiments, monocytes are further differentiated into M1 and / or M2 macrophages. iPSC-derived monocytes can be further differentiated into macrophages by exposure to MCSF for 3-4 days. The macrophages can be assessed for their ability to polarize, secrete pro- and anti-inflammatory cytokines, and for cytotoxic activity when co-cultured with cancer cells. M1 macrophages can be polarized with interferon gamma (IFN-γ, 50 ng / mL) and lipopolysaccharide (LPS, 10 ng / mL) for 48 hours, whereas M2 macrophages can be treated with IL-4 (10 ng / mL) for 48 hours. The efficiency of iPSC-derived monocytes differentiation into macrophages can be assessed by cell adherence, morphology, and surface marker expression (CD14, CD45, CD163). The ability of M1 and M2 polarized iPSC-derived macrophages to secrete TNFα, IL-12p70, and IL-10 can be assayed and compared to PBMC-derived macrophages. Finally, the ability of M1 and M2 polarized iPSC-derived macrophages to kill cancer cells can be assayed.

[0388] Cells can be reprogrammed by exposing them to specific extracellular cues and / or by ectopic expression of specific proteins, microRNAs, etc. While several reprogramming methods have been previously described, most that rely on ectopic expression require the introduction of exogenous DNA, which can carry mutation risks. DNA-free reprogramming methods based on direct delivery of reprogramming proteins have been reported. However, these methods are too inefficient and unreliable for commercial use. In addition, RNA-based reprogramming methods have been described (see, e.g., Angel. MIT Thesis. 2008. 1-56; Angel et al. PLoS ONE. 2010. 5, 107; Warren et al. Cell Stem Cell. 2010. 7, 618-630; Angel. MIT Thesis. 2011. 1-89; and Lee et al., Cell. 2012. 151, 547-558; the contents of all of which are hereby incorporated by reference). However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require many transfections (resulting in significant expense and opportunity for error), can reprogram only a limited number of cell types, can reprogram cells to only a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeders. The many drawbacks of previously disclosed RNA-based reprogramming methods make them undesirable for research, therapeutic or cosmetic use.

[0389] In some embodiments, reprogramming is performed by transfecting cells with one or more nucleic acids encoding one or more reprogramming factors, including, but not limited to Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In one embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a pluripotent stem cell. In another embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a glucose-responsive insulin-producing cell. Examples of other cells that can be reprogrammed and other cells to which a cell can be reprogrammed include, but are not limited to skin cells, pluripotent stem cells, MSCs, mesenchymal stromal / stem cells, β-cells, retinal pigmented epithelial cells, hematopoietic cells, hematopoietic stem cells, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells. In one embodiment, the cell is contacted with a medium that supports the reprogrammed cell. In one embodiment, the medium also supports the cell.

[0390] Importantly, infecting skin cells with viruses encoding Oct4, Sox2, Klf4, and c-Myc, combined with culturing the cells in a medium that supports the growth of cardiomyocytes, has been reported to cause reprogramming of the skin cells to cardiomyocytes, without first reprogramming the skin cells to pluripotent stem cells (See Efs et al Nat Cell Biol. 2011; 13:215-22, the contents of which are hereby incorporated by reference). In certain situations, direct reprogramming (reprogramming one somatic cell to another somatic cell without first reprogramming the somatic cell to a pluripotent stem cell, also known as “transdifferentiation”) may be desirable, in part because culturing pluripotent stem cells can be time-consuming and expensive, the additional handling involved in establishing and characterizing a stable pluripotent stem cell line can carry an increased risk of contamination, and the additional time in culture associated with first producing pluripotent stem cells can carry an increased risk of genomic instability and the acquisition of mutations, including point mutations, copy-number variations, and karyotypic abnormalities.

[0391] In embodiments, fewer total transfections may be required to reprogram a cell according to the methods of the present invention than according to other methods. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein from about 1 to about 12 transfections are performed during about 20 consecutive days, or from about 4 to about 10 transfections are performed during about 15 consecutive days, or from about 4 to about 8 transfections are performed during about 10 consecutive days. It is recognized that when a cell is contacted with a medium containing nucleic acid molecules, the cell may likely come into contact with and / or internalize more than one nucleic acid molecule either simultaneously or at different times. A cell can therefore be contacted with a nucleic acid more than once, e.g., repeatedly, even when a cell is contacted only once with a medium containing nucleic acids.

[0392] Of note, nucleic acids can contain one or more non-canonical or “modified” residues as described herein. For instance, any of the non-canonical nucleotides described herein can be used in the present reprogramming methods. In one embodiment, pseudouridine-5′-triphosphate can be substituted for uridine-5′-triphosphate in an in vitro-transcription reaction to yield synthetic RNA, wherein up to 100% of the uridine residues of the synthetic RNA may be replaced with pseudouridine residues. In vitro-transcription can yield RNA with residual immunogenicity, even when pseudouridine and 5-methylcytidine are completely substituted for uridine and cytidine, respectively (see, e.g., Angel. Reprogramming Human Somatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge, MA: MIT; 2011, the contents of which are hereby incorporated by reference). For this reason, it is common to add an immunosuppressant to the transfection medium when transfecting cells with RNA. In certain situations, adding an immunosuppressant to the transfection medium may not be desirable, in part because the recombinant immunosuppressant most commonly used for this purpose, B18R, can be expensive and difficult to manufacture. In one embodiment, the immunosuppressant is B18R or a biologically active fragment, analogue, variant or family-member thereof or dexamethasone or a derivative thereof. In one embodiment, the transfection medium does not contain an immunosuppressant, and the nucleic-acid dose is chosen to prevent excessive toxicity. In another embodiment, the nucleic-acid dose is less than about 1 mg / cm2 of tissue or less than about 1 mg / 100,000 cells or less than about 10 mg / kg.

[0393] In various cases, transfection of a cell with synthetic nucleic acids for reprogramming the cell may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV):where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In any of the herein-disclosed aspects or embodiments, a synthetic RNA molecule may be in the form of a circular RNA (circRNA). The circRNA are manufactured by methods do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA, the circRNA are manufactured by methods that do not require ribozymes to yield a circRNA, and / or the circRNA are manufactured by methods that do not require HPLC-based purification, e.g., post-ligation. A nucleic acid that can be manufactured into a circRNA has the structure: 5′-X-Y-A-IRES-B-CDS-C-Y′-Z 3′. Here, Y and Y′ each independently comprise one or more nucleotides and Y and Y′ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null. The CDS of a circRNA may encode one or more proteins of interest, the protein of interest being one or more reprogramming factors, optionally selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, Aicda, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof. In some cases, the CDS encodes two, three, four, five, six, seven, eight, nine, ten, eleven, or more reprogramming factor(s). Additional details regarding circRNAs useful in the present disclosure are described in PCT / US2022 / 026564, the contents of which are incorporated herein by reference in its entirety.

[0395] Reprogrammed cells produced according to certain embodiments of the present invention are suitable for therapeutic and / or cosmetic applications as they do not contain undesirable exogenous DNA sequences, and they are not exposed to animal-derived or human-derived products, which may be undefined, and which may contain toxic and / or pathogenic contaminants. Furthermore, the high speed, efficiency, and reliability of certain embodiments of the present invention may reduce the risk of acquisition and accumulation of mutations and other chromosomal abnormalities. Certain embodiments of the present invention can thus be used to generate cells that have a safety profile adequate for use in therapeutic and / or cosmetic applications. For example, reprogramming cells using RNA and the medium of the present invention, wherein the medium does not contain animal or human-derived components, can yield cells that have not been exposed to allogeneic material. Certain embodiments are therefore directed to a reprogrammed cell that has a desirable safety profile. In one embodiment, the reprogrammed cell has a normal karyotype. In another embodiment, the reprogrammed cell has fewer than about 5 copy-number variations (CNVs) relative to the patient genome, such as fewer than about 3 copy-number variations relative to the patient genome, or no copy-number variations relative to the patient genome. In yet another embodiment, the reprogrammed cell has a normal karyotype and fewer than about 100 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 50 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 10 single nucleotide variants in coding regions relative to the patient genome.

[0396] Endotoxins and nucleases can co-purify and / or become associated with other proteins, such as serum albumin. Recombinant proteins, in particular, can often have high levels of associated endotoxins and nucleases, due in part to the lysis of cells that can take place during their production. Endotoxins and nucleases can be reduced, removed, replaced or otherwise inactivated by many of the methods of the present invention, including, for example, by acetylation, by addition of a stabilizer such as sodium octanoate, followed by heat treatment, by the addition of nuclease inhibitors to the albumin solution and / or medium, by crystallization, by contacting with one or more ion-exchange resins, by contacting with charcoal, by preparative electrophoresis or by affinity chromatography. In embodiments, partially or completely reducing, removing, replacing, or otherwise inactivating endotoxins and / or nucleases from a medium and / or from one or more components of a medium is provided and this can increase the efficiency with which cells can be transfected and reprogrammed. Certain embodiments are therefore directed to a method for transfecting a cell with one or more nucleic acids, wherein the transfection medium is treated to partially or completely reduce, remove, replace or otherwise inactivate one or more endotoxins and / or nucleases. Other embodiments are directed to a medium that causes minimal degradation of nucleic acids. In one embodiment, the medium contains less than about 1 EU / mL, or less than about 0.1 EU / mL, or less than about 0.01 EU / mL.

[0397] In certain situations, protein-based lipid carriers such as serum albumin can be replaced with non-protein-based lipid carriers such as methyl-beta-cyclodextrin. The medium of the present invention can also be used without a lipid carrier, for example, when transfection is performed using a method that may not require or may not benefit from the presence of a lipid carrier, for example, using one or more lipid-based transfection reagents, polymer-based transfection reagents or peptide-based transfection reagents or using electroporation. Many protein-associated molecules, such as metals, can be highly toxic to cells in vivo. This toxicity can cause decreased viability, as well as the acquisition of mutations. Certain embodiments thus have the additional benefit of producing cells that are free from toxic molecules.

[0398] The associated-molecule component of a protein can be measured by suspending the protein in solution and measuring the conductivity of the solution. Certain embodiments are therefore directed to a medium that contains a protein, wherein about a 10% solution of the protein in water has a conductivity of less than about 500 μmho / cm. In one embodiment, the solution has a conductivity of less than about 50 μmho / cm. In another embodiment, less than about 0.65% of the dry weight of the protein comprises lipids and / or less than about 0.35% of the dry weight of the protein comprises free fatty acids.

[0399] Certain embodiments are therefore directed to a method for transfecting a cell with a nucleic acid, wherein the cell is transfected more than once, and wherein the amount of nucleic acid delivered to the cell is different for two of the transfections. In one embodiment, the cell proliferates between two of the transfections, and the amount of nucleic acid delivered to the cell is greater for the second of the two transfections than for the first of the two transfections. In another embodiment, the cell is transfected more than twice, and the amount of nucleic acid delivered to the cell is greater for the second of three transfections than for the first of the same three transfections, and the amount of nucleic acid delivered to the cells is greater for the third of the same three transfections than for the second of the same three transfections. In yet another embodiment, the cell is transfected more than once, and the maximum amount of nucleic acid delivered to the cell during each transfection is sufficiently low to yield at least about 80% viability for at least two consecutive transfections.

[0400] In embodiments, there are provided methods in which modulating the amount of nucleic acid delivered to a population of proliferating cells in a series of transfections can result in both an increased effect of the nucleic acid and increased viability of the cells. In embodiments, when cells are contacted with one or more nucleic acids encoding one or more reprogramming factors in a series of transfections, the efficiency of reprogramming can be increased when the amount of nucleic acid delivered in later transfections is greater than the amount of nucleic acid delivered in earlier transfections, for at least part of the series of transfections. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein one or more nucleic acids is repeatedly delivered to the cell in a series of transfections, and the amount of the nucleic acid delivered to the cell is greater for at least one later transfection than for at least one earlier transfection. In one embodiment, the cell is transfected from about 2 to about 10 times, or from about 3 to about 8 times, or from about 4 to about 6 times. In another embodiment, the one or more nucleic acids includes at least one RNA molecule, the cell is transfected from about 2 to about 10 times, and the amount of nucleic acid delivered to the cell in each transfection is the same as or greater than the amount of nucleic acid delivered to the cell in the most recent previous transfection. In yet another embodiment, the amount of nucleic acid delivered to the cell in the first transfection is from about 20 ng / cm2 to about 250 ng / cm2, or from 100 ng / cm2 to 600 ng / cm2. In yet another embodiment, the cell is transfected about 5 times at intervals of from about 12 to about 48 hours, and the amount of nucleic acid delivered to the cell is about 25 ng / cm2 for the first transfection, about 50 ng / cm2 for the second transfection, about 100 ng / cm2 for the third transfection, about 200 ng / cm2 for the fourth transfection, and about 400 ng / cm2 for the fifth transfection. In yet another embodiment, the cell is further transfected at least once after the fifth transfection, and the amount of nucleic acid delivered to the cell is about 400 ng / cm2.

[0401] Several molecules can be added to media by conditioning. Certain embodiments are therefore directed to a medium that is supplemented with one or more molecules that are present in a conditioned medium. In one embodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In another embodiment, the medium is supplemented with TGF-β or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, a cell is reprogrammed according to the method of the present invention, wherein the medium is not supplemented with TGF-β for from about 1 to about 5 days and is then supplemented with TGF-β for at least about 2 days. In yet another embodiment, the medium is supplemented with IL-6, IL-6R or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, the medium is supplemented with a sphingolipid or a fatty acid. In still another embodiment, the sphingolipid is lysophosphatidic acid, lysosphingomyelin, sphingosine-1-phosphate or a biologically active analogue, variant or derivative thereof.

[0402] In addition to mitotically inactivating cells, under certain conditions, irradiation can change the gene expression of cells, causing cells to produce less of certain proteins and more of certain other proteins than non-irradiated cells, for example, members of the Wnt family of proteins. In addition, certain members of the Wnt family of proteins can promote the growth and transformation of cells. In embodiments, the efficiency of reprogramming can be greatly increased by contacting a cell with a medium that is conditioned using irradiated feeders instead of mitomycin-c-treated feeders. In embodiments, the increase in reprogramming efficiency observed when using irradiated feeders is caused in part by Wnt proteins that are secreted by the feeders. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein the cell is contacted with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, family-member or agonist thereof, including agonists of downstream targets of Wnt proteins, and / or agents that mimic one or more of the biological effects of Wnt proteins, for example, 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

[0403] Because of the low efficiency of many DNA-based reprogramming methods, these methods may be difficult or impossible to use with cells derived from patient samples, which may contain only a small number of cells. In contrast, the high efficiency of certain embodiments of the present invention can allow reliable reprogramming of a small number of cells, including single cells. Certain embodiments are directed to a method for reprogramming a small number of cells. Other embodiments are directed to a method for reprogramming a single cell. In one embodiment, the cell is contacted with one or more enzymes. In another embodiment, the enzyme is collagenase. In yet another embodiment, the collagenase is animal-component free. In one embodiment, the collagenase is present at a concentration of from about 0.1 mg / mL to about 10 mg / mL, or from about 0.5 mg / mL to about 5 mg / mL. In another embodiment, the cell is a blood cell. In yet another embodiment, the cell is contacted with a medium containing one or more proteins that is derived from the patient's blood. In still another embodiment, the cell is contacted with a medium comprising: DMEM / F12+2 mM L-alanyl-L-glutamine+from about 5% to about 25% patient-derived serum, or from about 10% to about 20% patient-derived serum, or about 20% patient-derived serum.

[0404] In embodiments, transfecting cells with a mixture of RNA encoding Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof using the medium of the present invention can cause the rate of proliferation of the cells to increase. When the amount of RNA delivered to the cells is too low to ensure that all of the cells are transfected, only a fraction of the cells may show an increased proliferation rate. In certain situations, such as when generating a personalized therapeutic, increasing the proliferation rate of cells may be desirable, in part because doing so can reduce the time necessary to generate the therapeutic, and therefore can reduce the cost of the therapeutic. Certain embodiments are therefore directed to a method for transfecting a cell with a mixture of RNA encoding Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In one embodiment, the cell exhibits an increased proliferation rate. In another embodiment, the cell is reprogrammed.

[0405] While detailed examples are provided herein for the production of specific types of cells and for the production of therapeutics comprising specific types of cells, it is recognized that the methods of the present invention can be used to produce many other types of cells, and to produce therapeutics comprising one or more of many other types of cells, for example, by reprogramming a cell according to the methods of the present invention, and culturing the cell under conditions that mimic one or more aspects of development by providing conditions that resemble the conditions present in the cellular microenvironment during development.

[0406] Other embodiments are directed to a method for reprogramming a cell. In one embodiment, the cell is reprogrammed by contacting the cell with one or more nucleic acids. In one embodiment, the cell is contacted with a plurality of nucleic acids encoding at least one of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In another embodiment, the cell is contacted with a plurality of nucleic acids encoding a plurality of proteins including: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0407] Illustrative subjects or patients refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0408] In some embodiments, a synthetic RNA molecule is used to reprogram iPSCs into monocytes, which can then be further differentiated into M1 and / or M2 macrophages. In embodiments, the synthetic RNA molecule is mRNA. In embodiments, the synthetic RNA molecule is in vitro transcribed. In embodiments, the synthetic RNA is a circRNA.

[0409] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides. In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein, and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0410] In some embodiments, the synthetic RNA comprises a 5′ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a sequence that increases RNA stability in vivo, and the 5′-UTR optionally comprises an alpha-globin or beta-globin 5′-UTR. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a sequence that increases RNA stability in vivo, and the 3′-UTR optionally comprises an alpha-globin or beta-globin 3′-UTR. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0411] Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′-UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20 nt and about 250 nt or between about 120 nt and about 150 nt long. In a further embodiment, the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

[0412] In some embodiments, the RNA comprises a tail composed of a plurality of adenines with one or more guanines.

[0413] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0414] In embodiments, the one or more other nucleotides comprises deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0415] In embodiments, the one or more other nucleotides comprises deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0416] In embodiments, the one or more other nucleotides comprises deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0417] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues and deoxycytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region comprises fewer than 50% deoxyadenosine residues.

[0418] In embodiments, the one or more other nucleotides comprises guanosine residues.

[0419] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues. In embodiments, the tail region comprises more than 50% guanosine residues.

[0420] In embodiments, the one or more other nucleotides comprises cytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0421] In embodiments, the one or more other nucleotides comprises uridine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0422] In embodiments, the one or more other nucleotides comprise guanosine residues and cytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0423] In embodiments, the tail region comprises fewer than 50% adenosine residues.

[0424] In embodiments, the tail is (A)150 (SEQ ID NO: 61). In embodiments, the tail is (A39G)3(A)30 (SEQ ID NO: 62). In embodiments, the tail is (A19G)7(A)10 (SEQ ID NO: 63). In embodiments, the tail is (A9G)15 (SEQ ID NO: 64).

[0425] In embodiments, the length of the tail region is between about 80 nucleotides and about 120 nucleotides, about 120 nucleotides and about 160 nucleotides, about 160 nucleotides and about 200 nucleotides, about 200 nucleotides and about 240 nucleotides, about 240 nucleotides and about 280 nucleotides, or about 280 nucleotides and about 320 nucleotides.

[0426] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0427] In embodiments, the RNA comprises a 5′ cap structure. In embodiments, the RNA 5′-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5′-UTR comprises a sequence that increases RNA stability in vivo, and the 5′-UTR may comprise an alpha-globin or beta-globin 5′-UTR.

[0428] In embodiments, the RNA 3′-UTR comprises a sequence that increases RNA stability in vivo, and the 3′-UTR may comprise an alpha-globin or beta-globin 3′-UTR. In embodiments, the RNA comprises a 3′ poly(A) tail. In embodiments, the RNA 3′ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0429] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0430] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is synthetic.

[0431] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides.

[0432] In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0433] Further description of reprogramming is found in one or more of WO / 2013 / 086008, WO / 2014 / 071219, WO / 2015 / 117021, WO / 2016 / 131052, WO / 2018 / 035377, WO / 2019 / 191341, WO / 2021 / 003462, WO2021 / 231549, or WO2021 / 222389. The entire contents of which are incorporated by reference in their entirety.Gene-Editing

[0434] In any herein disclosed aspect or embodiment, a cell is gene-edited.

[0435] In embodiments, gene-editing a cell comprises contacting the cell with a synthetic nucleic acid encoding one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR / Cas9, Cas9, xCas9, Cas12a(Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyz (SEQ ID NO: 16), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.

[0436] In embodiments, the gene-editing protein comprises: (i) a DNA-binding domain comprising a plurality of repeat sequences and (ii) the nuclease domain comprising a catalytic domain of a nuclease. In embodiments, the at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 22) and is optionally between 36 and 39 amino acids long, where:

[0437] v is Q, D or E,

[0438] w is S or N,

[0439] x is I, H, N, or I,

[0440] y is D, A, I, N, H, K, S, G, or null,

[0441] z is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), and

[0442] α is four consecutive amino acids.

[0443] In embodiments, α comprises at least one glycine (G) residue. In embodiments, α comprises at least one histidine (H) residue. In embodiments, α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35. In embodiments, α comprises at least one aspartic acid (D) residue. In embodiments, α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.

[0444] In embodiments, α comprises one or more hydrophilic residues, optionally selected from: a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K); a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C); a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).

[0445] In some embodiments, α comprises one or more polar and positively charged hydrophilic amino acids selected from arginine (R) and lysine (K). In some embodiments, α comprises one or more polar and neutral of charge hydrophilic amino acids selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In some embodiments, α comprises one or more polar and negatively charged hydrophilic amino acids selected from aspartate (D) and glutamate (E). In some embodiments, α comprises one or more aromatic, polar and positively charged hydrophilic amino acids selected from histidine (H).

[0446] In embodiments, α comprises one or more hydrophobic residues, optionally selected from: a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In some embodiments, α comprises one or more hydrophobic, aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In some embodiments, α comprises one or more aromatic amino acids selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In embodiments, the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.

[0447] In embodiments, α is selected from GHGG (SEQ ID NO: 31), HGSG (SEQ ID NO: 32), HGGG (SEQ ID NO: 33), from GGHD (SEQ ID NO: 34), GAHD (SEQ ID NO: 35), AHDG (SEQ ID NO: 36), PHDG (SEQ ID NO: 37), GPHD (SEQ ID NO: 38), GHGP (SEQ ID NO: 39), PHGG (SEQ ID NO: 40), PHGP (SEQ ID NO: 41), AHGA (SEQ ID NO: 42), LHGA (SEQ ID NO: 43), VHGA (SEQ ID NO: 44), IVHG (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), RHGD (SEQ ID NO: 47), RDHG (SEQ ID NO: 48), RHGE (SEQ ID NO: 49), HRGE (SEQ ID NO: 50), RHGD (SEQ ID NO: 47), HRGD (SEQ ID NO: 51), GPYE (SEQ ID NO: 52), NHGG (SEQ ID NO: 53), THGG (SEQ ID NO: 54), GTHG (SEQ ID NO: 21), GSGS (SEQ ID NO: 56), GSGG (SEQ ID NO: 57), GGGG (SEQ ID NO: 58), GRGG (SEQ ID NO: 59), and GKGG (SEQ ID NO: 4260

[0448] In embodiments, the gene-editing protein has a DNA binding domain having at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (SEQ ID NO: 65; the “*RVD*” corresponds to the dinucleotide “xy” of SEQ ID NO: 22).

[0449] In embodiments, the repeat sequence is 33 or 34 amino acids long. In embodiments, the repeat sequence is 36-39 amino acids long. In some embodiments, the repeat sequence is 36 amino acids long. In some embodiments, the repeat sequence is 37 amino acids long. In some embodiments, the repeat sequence is 38 amino acids long. In some embodiments, the repeat sequence is 39 amino acids long.

[0450] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17) or LTPvQVVAIAwxyzGTHG (SEQ ID NO: 18) and is from 36 to 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20) and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In some embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C.

[0451] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 22) and is from 36 to 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), a is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, a is selected from GHGG (SEQ ID NO: 31), HGSG (SEQ ID NO: 32), HGGG (SEQ ID NO: 33), GGHD (SEQ ID NO: 34), GAHD (SEQ ID NO: 35), AHDG (SEQ ID NO: 36), PHDG (SEQ ID NO: 37), GPHD (SEQ ID NO: 38), GHGP (SEQ ID NO: 39), PHGG (SEQ ID NO: 40), PHGP (SEQ ID NO: 41), AHGA (SEQ ID NO: 42), LHGA (SEQ ID NO: 43), VHGA (SEQ ID NO: 44), IVHG (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), RHGD (SEQ ID NO: 47), RDHG (SEQ ID NO: 48), RHGE (SEQ ID NO: 49), HRGE (SEQ ID NO: 50), RHGD (SEQ ID NO: 47), HRGD (SEQ ID NO: 51), GPYE (SEQ ID NO: 52), NHGG (SEQ ID NO: 53), THGG (SEQ ID NO: 54), GTHG (SEQ ID NO: 21), GSGS (SEQ ID NO: 56), GSGG (SEQ ID NO: 57), GGGG (SEQ ID NO: 58), GRGG (SEQ ID NO: 59), and GKGG (SEQ ID NO: 60). In some embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C. Certain embodiments are directed to a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising a first region that recognizes a predetermined nucleotide sequence and a second region with endonuclease activity, wherein the first region contains an artificial TAL effector repeat domain comprising one or more repeat units about 36 amino acids in length which differ from each other by no more than seven amino acids, and wherein the repeat domain is engineered for recognition of the predetermined nucleotide sequence. In one embodiment, the first region contains the amino acid sequence: LTPXQVVAIAS (SEQ ID NO: 29) where X can be either E or Q. In another embodiment, the amino acid sequence LTPXQVVAIAS (SEQ ID NO: 29) of the encoded non-naturally occurring fusion protein is immediately followed by an amino acid sequence selected from: HD, NG, NS, NI, NN, and N. In a further embodiment, the fusion protein comprises restriction endonuclease activity.

[0452] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzHG, (SEQ ID NO: 30) wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG, (SEQ ID NO: 30) wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG, (SEQ ID NO: 30) wherein “v” is D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAIwyzHG (SEQ ID NO: 55), wherein “v” is D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzHG (SEQ ID NO: 66), wherein “v” is D or E, “w” is S or N, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 30), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 23), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 30), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx (SEQ ID NO: 67), wherein “v” is D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy (SEQ ID NO: 16), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwyzGHGG (SEQ ID NO: 70), wherein “v” is Q, D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzGHGG, (SEQ ID NO: 71) wherein “v” is Q, D or E, “w” is S or N, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, V is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx (SEQ ID NO: 67), wherein “v” is Q, D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy (SEQ ID NO: 72), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G.

[0453] The above-mentioned gene-editing proteins comprise a repeat variable di-residue (RVD) at residue 12 or 13, e.g., at “x” and “y” in the various above-mentioned repeat sequences, e.g., LTPvQVVAIAwxyzα (SEQ ID NO: 22), which targets the DNA-binding domain to a target DNA molecule. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(null), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(null), and IG.

[0454] In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HD. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is N(null). In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HA. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is ND. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HI. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NN. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NH. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NK. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is HN. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NA. In some embodiments, the RVD recognizing an A residue in the nucleic acid molecule is Nl. In some embodiments, the RVD recognizing an A residue in the nucleic acid molecule is NS. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is NG. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is HG. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is H(null). In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is IG.

[0455] In some embodiments, alternative DNA binding domains are employed.

[0456] For example, the alternative DNA binding domains described herein are, in embodiments, paired with the novel engineered nuclease domains described herein.

[0457] For example, the alternative DNA binding domains described herein are, in embodiments, used in the conditional activity: temperature dependence methods described herein.

[0458] For example, the alternative DNA binding domains described herein are, in embodiments, used in the conditional activity: methylation status methods described herein.

[0459] In embodiments, the engineered gene-editing proteins do not require a thymine (T) in the zero position of the target site (“To”).

[0460] In embodiments, the engineered gene-editing proteins that comprise DNA-binding domains comprise alterations in the in the N-terminal region to remove the T0 requirement.

[0461] In embodiments, there is provided a method of gene-editing a cell with one or more of the present gene-editing proteins, optionally with also using a linear DNA repair template, optionally also using conditional activity methods described herein, where the target site lacks a thymine (T) in the zero position.

[0462] Wild type N-terminal region is characterized by the sequence: Asp225-IVGVGKQWSGARAL-Glu240 (DIVGVGKQWSGARALE; SEQ ID NO: 73). In embodiments, there is provided the engineered N-terminal region of Asp225-IVGVGKQKRGARAL-Glu240 (underling showing the change WS->KR) (DIVGVGKQKRGARALE; SEQ ID NO: 74).

[0463] In embodiments, there is provided an engineered N-terminal region in which KQWS is replaced with one or more amino acids, e.g., about 2-10 amino acids, or about 4-10 amino acids, or about 6-10 amino acids, or about 8-10 amino acids, or about 4 amino acids, or about 6 amino acids, or about 8 amino acids, or about 10 amino acids.

[0464] In embodiments, there is provided the engineered N-terminal region of Asp225-IVGVGGSKRGAGSGARAL-Glu244 (underling showing the change KQWS->GSKRGAGS) (DIVGVGGSKRGAGSGARALE; SEQ ID NO: 75).

[0465] In some cases, a cell is contacted with a demethylating agent during the process of gene-editing. In embodiments, the demethylating agent is selected from 5-azacitidine and 5-aza-2′-deoxycitidine (decitabine).

[0466] In some embodiments, the gene-editing protein comprises: (a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13; and (b) the nuclease domain comprising a catalytic domain, the catalytic domain comprising a hybrid of the catalytic domains of Fokl and Stsl, comprising the α1, α2, α3, α4, α5, α6, β1, β2, β, β4, β5, and β6 domains of Fokl with at least one of the domains of Fokl being substituted in whole or in part with the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Stsl and optionally comprising at least one mutation. In embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

[0467] In some embodiments, certain fragments of an endonuclease cleavage domain are used, including fragments that are truncated at the N-terminus, fragments that are truncated at the C-terminus, fragments that have internal deletions, and fragments that combine N-terminus, C-terminus, and / or internal deletions, which maintain part or all of the catalytic activity of the full endonuclease cleavage domain. Determining whether a fragment can maintain part, or all of the catalytic activity of the full domain can be accomplished by, for example, synthesizing a gene-editing protein that contains the fragment according to the methods of the present invention, inducing cells to express the gene-editing protein according to the methods of the present invention, and measuring the efficiency of gene editing. In some embodiments, a measurement of gene-editing efficiency is used to ascertain whether any specific fragment maintains part or all of the catalytic activity of the full endonuclease cleavage domain. Certain embodiments are therefore directed to a biologically active fragment of an endonuclease cleavage domain. In one embodiment, the endonuclease cleavage domain is selected from: FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineered variant or biologically active fragment thereof, or a hybrid or chimera thereof.

[0468] In embodiments, the gene-editing protein comprises a linker. In another embodiment, the linker connects a DNA-binding domain to a nuclease domain. In a further embodiment, the linker is between about 1 and about 10 amino acids long. In some embodiments, the linker is about 1, about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids long. In one embodiment, the gene-editing protein is capable of generating a nick or a double-strand break in a target DNA molecule.

[0469] In embodiments, the gene-editing protein is any of those described in International Patent Publication No. WO 2014 / 071219 or WO2021 / 231549, hereby incorporated by reference in their entireties.

[0470] In various embodiments, the cell is transfected (e.g., contacted) with a synthetic nucleic acid encoding the gene-editing protein at about 30° C. to about 35° C., e.g., without limitation about 33° C. In embodiments, the contacting occurs at about 30° C. In some embodiments, the contacting occurs at about 31° C. In some embodiments, the contacting occurs at about 32° C. In some embodiments, the contacting occurs at about 33° C. In some embodiments, the contacting occurs at about 34° C. In some embodiments, the contacting occurs at about 35° C. In embodiments, the gene-editing protein is functionally temperature-switchable. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. to about 35° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 31° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 32° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 33° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 34° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 35° C.

[0471] Further description of temperature-sensitive gene-editing is found in WO2021 / 231549. The entire contents of which are incorporated by reference in their entirety.

[0472] In embodiments, the synthetic nucleic acid encoding the gene-editing protein is transfected along with a repair template. In some cases, the repair template is a double stranded synthetic oligodeoxynucleotide (dsODNs). In embodiments, the dsODNs comprises a repair template and comprises the TTAGGG motif. In some cases, the dsODN comprises a repair template and lacks the TTAGGG motif and a separate dsODNs comprising the TTAGGG motif is transfected into the cell. In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides. In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein, and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0473] In some embodiments, the synthetic RNA comprises a 5′ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a sequence that increases RNA stability in vivo, and the 5′-UTR optionally comprises an alpha-globin or beta-globin 5′-UTR. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a sequence that increases RNA stability in vivo, and the 3′-UTR optionally comprises an alpha-globin or beta-globin 3′-UTR. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0474] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0475] In various cases, transfection of a cell with synthetic nucleic acids for gene-editing the cell may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In any of the herein-disclosed aspects or embodiments, a synthetic RNA molecule encoding the gene-editing protein may be in the form of a circular RNA (circRNA). The circRNA are manufactured by methods do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA, the circRNA are manufactured by methods that do not require ribozymes to yield a circRNA, and / or the circRNA are manufactured by methods that do not require HPLC-based purification, e.g., post-ligation. A nucleic acid that can be manufactured into a circRNA has the structure: 5′-X-Y-A-IRES-B-CDS-C-Y′-Z 3′. Here, Y and Y′ each independently comprise one or more nucleotides and Y and Y′ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null. The CDS of a circRNA encodes the gene-editing protein(s). Additional details regarding circRNAs useful in the present disclosure are described in PCT / US2022 / 026564, the contents of which are incorporated herein by reference in its entirety.

[0477] In some embodiments, the synthetic RNA comprises a 5′ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a sequence that increases RNA stability in vivo, and the 5′-UTR optionally comprises an alpha-globin or beta-globin 5′-UTR. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a sequence that increases RNA stability in vivo, and the 3′-UTR optionally comprises an alpha-globin or beta-globin 3′-UTR. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0478] Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′-UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20 nt and about 250 nt or between about 120 nt and about 150 nt long. In a further embodiment, the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

[0479] In some embodiments, the RNA comprises a tail composed of a plurality of adenines with one or more guanines.

[0480] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0481] In embodiments, the one or more other nucleotides comprises deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0482] In embodiments, the one or more other nucleotides comprises deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0483] In embodiments, the one or more other nucleotides comprises deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0484] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues and deoxycytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region comprises fewer than 50% deoxyadenosine residues.

[0485] In embodiments, the one or more other nucleotides comprises guanosine residues.

[0486] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues. In embodiments, the tail region comprises more than 50% guanosine residues.

[0487] In embodiments, the one or more other nucleotides comprises cytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0488] In embodiments, the one or more other nucleotides comprises uridine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0489] In embodiments, the one or more other nucleotides comprise guanosine residues and cytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0490] In embodiments, the tail region comprises fewer than 50% adenosine residues.

[0491] In embodiments, the tail is (A)150 (SEQ ID NO: 61). In embodiments, the tail is (A39G)3(A)30 (SEQ ID NO: 62). In embodiments, the tail is (A19G)7(A)10 (SEQ ID NO: 63). In embodiments, the tail is (A9G)15 (SEQ ID NO: 64).

[0492] In embodiments, the length of the tail region is between about 80 nucleotides and about 120 nucleotides, about 120 nucleotides and about 160 nucleotides, about 160 nucleotides and about 200 nucleotides, about 200 nucleotides and about 240 nucleotides, about 240 nucleotides and about 280 nucleotides, or about 280 nucleotides and about 320 nucleotides.

[0493] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0494] In embodiments, the RNA comprises a 5′ cap structure. In embodiments, the RNA 5′-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5′-UTR comprises a sequence that increases RNA stability in vivo, and the 5′-UTR may comprise an alpha-globin or beta-globin 5′-UTR.

[0495] In embodiments, the RNA 3′-UTR comprises a sequence that increases RNA stability in vivo, and the 3′-UTR may comprise an alpha-globin or beta-globin 3′-UTR. In embodiments, the RNA comprises a 3′ poly(A) tail. In embodiments, the RNA 3′ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0496] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0497] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is synthetic. In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0498] Further description of gene-editing is found in one or more of WO / 2013 / 086008, WO / 2014 / 071219, WO / 2015 / 117021, WO / 2016 / 131052, WO / 2018 / 035377, WO / 2019 / 191341, WO / 2021 / 003462, WO2021 / 231549, or WO2021 / 222389. The entire contents of which are incorporated by reference in their entirety.RNA Modifications

[0499] In embodiments, the present disclosure relates to RNA-based modifications, e.g., reprogramming and / or gene-editing. In some embodiments, an RNA molecule encodes a gene-editing protein. In some embodiments, a RNA molecule encodes a reprogramming factor.

[0500] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides.

[0501] In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein, and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides.

[0502] In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0503] In embodiments, the synthetic RNA molecule is mRNA comprising one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0504] In some embodiments, the one or more non-canonical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine. In some embodiments, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the non-canonical nucleotides are one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0505] In some embodiments, at least about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of cytidine residues are non-canonical nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine.

[0506] In some embodiments, at least about 20%, or about 30%, or about 40%, or about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of uridine residues are non-canonical nucleotides selected from 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0507] In some embodiments, at least about 10% (e.g., 10%, or about 20%, or about 30%, or about 40%, or about 50%) of guanosine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the RNA contains no more than about 50% 7-deazaguanosine in place of guanosine residues.

[0508] In some embodiments, the synthetic RNA molecule does not contain non-canonical nucleotides in place of adenosine residues.

[0509] Other non-canonical nucleotides that can be used in place of or in combination with 5-methyluridine include but are not limited to: pseudouridine and 5-methylpseudouridine (a.k.a. “1-methylpseudouridine”, a.k.a. “N1-methylpseudouridine”) or one or more derivatives thereof. Other non-canonical nucleotides that can be used in place of or in combination with 5-methylcytidine and / or 5-hydroxymethylcytidine include, but are not limited to: pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, N4-acetylcytidine or one or more derivatives thereof. In certain embodiments, for example, when performing only a single transfection or when the cells being transfected are not particularly sensitive to transfection-associated toxicity or innate-immune signaling, the fractions of non-canonical nucleotides can be reduced. Reducing the fraction of non-canonical nucleotides can be beneficial, in part, because reducing the fraction of non-canonical nucleotides can reduce the cost of the nucleic acid. In certain situations, for example, when minimal immunogenicity of the nucleic acid is desired, the fractions of non-canonical nucleotides can be increased.

[0510] Note that alternative naming schemes exist for certain non-canonical nucleotides. For example, in certain situations, 5-methylpseudouridine can be referred to as “3-methylpseudouridine” or “N3-methylpseudouridine” or “1-methylpseudouridine” or “N1-methylpseudouridine”. Nucleotides that contain the prefix “amino” can refer to any nucleotide that contains a nitrogen atom bound to the atom at the stated position of the nucleotide, for example, 5-aminocytidine can refer to 5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly, nucleotides that contain the prefix “methyl” can refer to any nucleotide that contains a carbon atom bound to the atom at the stated position of the nucleotide, for example, 5-methylcytidine can refer to 5-methylcytidine, 5-ethylcytidine, and 5-hydroxymethylcytidine, nucleotides that contain the prefix “thio” can refer to any nucleotide that contains a sulfur atom bound to the atom at the given position of the nucleotide, and nucleotides that contain the prefix “hydroxy” can refer to any nucleotide that contains an oxygen atom bound to the atom at the given position of the nucleotide, for example, 5-hydroxyuridine can refer to 5-hydroxyuridine and uridine with a methyl group bound to an oxygen atom, wherein the oxygen atom is bound to the atom at the 5C position of the uridine.

[0511] In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the nucleic acid comprises one or more non-canonical nucleotide members of the 5 methylcytidine de-methylation pathway. In some embodiments, the nucleic acid comprises at least one of: 5 methylcytidine, 5 hydroxymethylcytidine, 5 formylcytidine, and 5 carboxycytidine or a derivative thereof. In some embodiments, the nucleic acid comprises at least one of: pseudouridine, 5 methylpseudouridine, 5 methyluridine, 5 methylcytidine, 5 hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof.

[0512] Certain non-canonical nucleotides can be incorporated more efficiently than other non-canonical nucleotides into RNA molecules by RNA polymerases that are commonly used for in vitro transcription, due in part to the tendency of these certain non-canonical nucleotides to participate in standard base-pairing interactions and base-stacking interactions, and to interact with the RNA polymerase in a manner similar to that in which the corresponding canonical nucleotide interacts with the RNA polymerase. As a result, certain nucleotide mixtures containing one or more non-canonical nucleotides can be beneficial in part because in vitro-transcription reactions containing these nucleotide mixtures can yield a large quantity of RNA. Certain embodiments are therefore directed to a nucleotide mixture containing one or more nucleotides that includes one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine. Nucleotide mixtures include, but are not limited to (numbers preceding each nucleotide indicate an exemplary fraction of the non-canonical nucleotide triphosphate in an in vitro-transcription reaction, for example, 0.2 pseudoisocytidine refers to a reaction containing adenosine-5′-triphosphate, guanosine-5′-triphosphate, uridine-5′-triphosphate, cytidine-5′-triphosphate, and pseudoisocytidine-5′-triphosphate, wherein pseudoisocytidine-5′-triphosphate is present in the reaction at an amount approximately equal to 0.2 times the total amount of pseudoisocytidine-5′-triphosphate+cytidine-5′-triphosphate that is present in the reaction, with amounts measured either on a molar or mass basis, and wherein more than one number preceding a nucleoside indicates a range of exemplary fractions): 1.0 pseudouridine, 0.1-0.8 2-thiouridine, 0.1-0.8 5-methyluridine, 0.2-1.0 5-hydroxyuridine, 0.2-1.0 5-methoxyuridine, 0.1-1.0 5-aminouridine, 0.1-1.0 4-thiouridine, 0.1-1.0 2-thiopseudouridine, 0.1-1.0 4-thiopseudouridine, 0.1-1.0 5-hydroxypseudouridine, 0.2-1 5-methylpseudouridine, 0.2-1.0 5-methoxypseudouridine, 0.1-1.0 5-aminopseudouridine, 0.2-1.0 2-thiocytidine, 0.1-0.8 pseudoisocytidine, 0.2-1.0 5-methylcytidine, 0.2-1.0 5-hydroxycytidine, 0.2-1.0 5-hydroxymethylcytidine, 0.2-1.0 5-methoxycytidine, 0.1-1.0 5-aminocytidine, 0.2-1.0 N4-methylcytidine, 0.2-1.0 5-methylpseudoisocytidine, 0.2-1.0 5-hydroxypseudoisocytidine, 0.2-1.0 5-aminopseudoisocytidine, 0.2-1.0 N4-methylpseudoisocytidine, 0.2-1.0 2-thiopseudoisocytidine, 0.2-1.0 7-deazaguanosine, 0.2-1.0 6-thioguanosine, 0.2-1.0 6-thio-7-deazaguanosine, 0.2-1.0 8-azaguanosine, 0.2-1.0 7-deaza-8-azaguanosine, 0.2-1.0 6-thio-8-azaguanosine, 0.1-0.5 7-deazaadenosine, and 0.1-0.5 N6-methyladenosine.

[0513] In some embodiments, the RNA comprising one or more non-canonical nucleotides composition or synthetic polynucleotide composition (e.g., which may be prepared by in vitro transcription) contains substantially or entirely the canonical nucleotide at positions having adenine or “A” in the genetic code. The term “substantially” in this context refers to at least 90%. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) 7-deazaguanosine at positions with “G” in the genetic code as well as the corresponding canonical nucleotide “G”, and the canonical and non-canonical nucleotide at positions with G may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three or four) of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine at positions with “C” in the genetic code as well as the canonical nucleotide “C”, and the canonical and non-canonical nucleotide at positions with C may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “C” are as described in the preceding paragraph. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three, or four) of 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine at positions with “U” in the genetic code as well as the canonical nucleotide “U”, and the canonical and non-canonical nucleotide at positions with “U” may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “U” are as described in the preceding paragraph.

[0514] In embodiments, combining certain non-canonical nucleotides can be beneficial in part because the contribution of non-canonical nucleotides to lowering the toxicity of RNA molecules can be additive. Certain embodiments are therefore directed to a nucleotide mixture, wherein the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, for example, the nucleotide mixture contains both pseudoisocytidine and 7-deazaguanosine or the nucleotide mixture contains both N4-methylcytidine and 7-deazaguanosine, etc. In one embodiment, the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, and each of the non-canonical nucleotides is present in the mixture at the fraction listed above, for example, the nucleotide mixture contains 0.1-0.8 pseudoisocytidine and 0.2-1.0 7-deazaguanosine or the nucleotide mixture contains 0.2-1.0 N4-methylcytidine and 0.2-1.0 7-deazaguanosine, etc.

[0515] In certain situations, for example, when it may not be necessary or desirable to maximize the yield of an in vitro-transcription reaction, nucleotide fractions other than those given above may be used. The exemplary fractions and ranges of fractions listed above relate to nucleotide-triphosphate solutions of typical purity (greater than 90% purity). Larger fractions of these and other nucleotides can be used by using nucleotide-triphosphate solutions of greater purity, for example, greater than about 95% purity or greater than about 98% purity or greater than about 99% purity or greater than about 99.5% purity, which can be achieved, for example, by purifying the nucleotide triphosphate solution using existing chemical-purification technologies such as high-pressure liquid chromatography (HPLC) or by other means. In one embodiment, nucleotides with multiple isomers are purified to enrich the desired isomer.

[0516] In some embodiments, the one or more non-canonical nucleotides avoids substantial cellular toxicity.

[0517] In some embodiments, the non-canonical nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine, optionally at an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-canonical nucleotides.

[0518] In some embodiments, at least about 50% of cytidine residues are non-canonical nucleotides, and which are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0519] In some embodiments, at least about 75% or at least about 90% of cytidine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0520] In some embodiments, at least about 20% of uridine, or at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0521] In some embodiments, at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0522] In some embodiments, at least about 10% of guanine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the synthetic RNA comprises no more than about 50% 7-deazaguanosine in place of guanosine residues. In some embodiments, the synthetic RNA does not comprise non-canonical nucleotides in place of adenosine residues.Methods of Treatment

[0523] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0524] In various embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a chimeric antigen receptor (CAR). In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the CAR inserts into the break. In these embodiments, the cell in the subject expresses the CAR.

[0525] In numerous embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a cytokine. In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the cytokine inserts into the break. In these embodiments, the cell in the subject expresses or over expresses the cytokine.

[0526] When the synthetic mRNA and / or the repair template is administered to a subject, the synthetic mRNA and / or the repair is combined with a lipid system comprising a compound of Formula (IV).

[0527] In these embodiments, the cytotoxic lymphocyte targets and kills cancer cells and the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0528] In various embodiments, the present methods and compositions find use in methods of treating, preventing, or ameliorating a disease, disorder, and / or condition. For instance, in some embodiments, the described methods of in vivo delivery, including administration strategies, and formulations are used in a method of treatment. In some methods, the described methods reduce symptoms associated with a disease. In some embodiments, the methods eliminate the underlying cause of the disease. In some embodiments, the methods are used in the treatment of a disease requiring immunosuppression. In some embodiments, the methods reduce inflammation. In some embodiments, the methods reduce immune response.

[0529] In various embodiments, the present invention pertains to pharmaceutical compositions comprising the recombinantly engineered cells described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the present invention pertains to pharmaceutical compositions comprising the iPSC-derived cells of the lymphoid lineage, including cytotoxic lymphocytes, iPSC-derived cells of the myeloid lineage, e.g., monocytes which can be differentiated into functional M1 and M2 macrophages having enhanced cytokine secretion and tumor cell-killing activity, and / or synthetic RNA molecules encoding the gene-editing protein or expression cassettes for expressing a protein of interest, e.g., a CAR or for expressing or overexpressing a cytokine.

[0530] In embodiments, the disclosed composition is suitable for use in the treatment of amyotrophic lateral sclerosis (ALS), spinal cord injury, degenerative disc disease, coronary artery disease, acute myocardial infarction, alcoholic liver cirrhosis, hepatitis C virus (HCV)-induced cirrhosis, multiple sclerosis (MS), osteoarthritis (OA), osteoarthritis of the knee, kidney allograft, critical limb ischemia, ischemic cardiomyopathy, Crohn's disease, idiopathic pulmonary fibrosis, anal fistula, spinal cord injury, systemic lupus erythematosus (SLE), acute respiratory distress syndrome (ARDS), acute graft-versus-host disease (aGvHD), preterm bronchopulmonary dysplasia (BPD), autism nonischemic heart failure, and / or Type 2 diabetes mellitus.

[0531] In embodiments, the present methods relate to therapeutic use in autoimmune diseases or disorders. Examples of autoimmune diseases or disorders that may be treated or prevented by the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), irritable bowel disease (IBD), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis. Preferably autoimmune disorders that may be treated or prevented by the present compositions include rheumatoid arthritis, type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, and atopy.

[0532] In embodiments, the present methods relate to therapeutic use in degenerative diseases or disorders. A degenerative diseases or disorders is a disease in which the function or structure of the affected tissues or organs will progressively deteriorate overtime. Examples of degenerative diseases that can be treated or prevented with the present invention include Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Parkinson's Disease, Multiple system atrophy, Niemann Pick disease, Atherosclerosis, Progressive supranuclear palsy, Tay-Sachs Disease, Diabetes, Heart Disease, Keratoconus, Inflammatory Bowel Disease (IBD), Prostatitis, Osteoarthritis, Osteoporosis, Rheumatoid Arthritis, Huntington's Disease, Chronic traumatic encephalopathy, Epilepsy, Dementia, Renal failure, Multiple sclerosis, Malaria with CNS degeneration, Neuro-AIDS, Lysosomal storage diseases, Encephalitis of viral, bacterial or autoimmune origin.

[0533] In embodiments, the present methods relate to therapeutic use in a lung diseases or disorders.

[0534] In embodiments, the lung disease or disorder is a lung disease or disorder that would benefit therapeutically from suppression of immune responses in the lung. In some embodiments, inflammation is associated with the lung disease or disorder.

[0535] In some embodiments, the lung disease or disorder is selected from Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD), Common Cold, Croup, Cystic Fibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis, Influenza, Lung Cancer, Pandemic Flu, Pertussis, Pleurisy, Pneumonia, Pulmonary Embolism, Pulmonary Hypertension, Respiratory Syncytial Virus (RSV), Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS), and Tuberculosis.

[0536] In some embodiments, the lung disease or disorder is chronic obstructive pulmonary disease (COPD), reactive airway disease such as asthma, bronchiolitis, acute lung injury, lung allograft rejection (acute or chronic), pulmonary fibrosis, interstitial lung disease or hypersensitivity pneumonitis. In embodiments, the disease or disorder is an acute lung injury (ALI). In embodiments, the ALI is a pulmonary disorder that can be induced directly by inhalation of chemicals (chemical induced acute lung injury) or other means (e.g., infection) or can be induced indirectly by systemic injury (e.g., infection). Acute lung injury includes subcategories of respiratory distress syndromes including infant respiratory distress syndrome (IRDS), hyaline membrane disease (HMD), neonatal respiratory distress syndrome (NRDS), respiratory distress syndrome of newborn (RDSN), surfactant deficiency disorder (SDD), acute respiratory distress syndrome (ARDS), respiratory complication from systemic inflammatory response syndrome (SIRS), or severe acute respiratory syndrome (SARS).

[0537] In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms associated with a viral infection.

[0538] In embodiments, the composition is suitable for use in the treatment of an infectious disease, optionally selected from an infection with a pathogen, optionally selected from a bacterium, virus, fungus, or parasite.

[0539] In embodiments, the pathogen is a virus. In embodiments, the virus is: (a) an influenza virus, optionally selected from Type A, Type B, Type C, and Type D influenza viruses, or (b) a member of the Coronaviridae family, optionally selected from (i) a betacoronavirus, optionally selected from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43 or (ii) an alphacoronavirus, optionally selected from HCoV-NL63 and HCoV-229E.

[0540] In embodiments, the virus is SARS-CoV-2. In embodiments, the virus is SARS-CoV-2, which has caused COVID-19. In embodiments, the COVID-19 is characterized by one or more of fever, cough, shortness of breath, diarrhea, upper respiratory symptoms, lower respiratory symptoms, pneumonia, and respiratory distress.

[0541] In some embodiments, the composition is suitable for use in the treatment of an infection, wherein the infection is a coronavirus infection. In some embodiments, the coronavirus infection is one or more of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E. In various embodiments, the coronavirus infection is SARS or COVID-19. In further embodiments, the subject is infected by SARS-CoV-2.

[0542] In embodiments, the therapy prevents or mitigates development of acute respiratory distress syndrome (ARDS) in a patient when administered. In embodiments, the therapy improves oxygenation in a patient when administered. In embodiments, the therapy improves systemic blood pressure oxygenation in a patient when administered, e.g., reducing or mitigating shock, e.g., requiring less pressor support. In embodiments, the therapy improves lung and / or alveolar permeability in a patient when administered.

[0543] In embodiments, the therapy prevents or mitigates a transition from respiratory distress to cytokine imbalance in a patient when administered. In embodiments, the therapy reverses or prevents a cytokine storm in a patient when administered. In embodiments, the therapy reverses or prevents a cytokine storm in the lungs or systemically in a patient when administered. In embodiments, the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis.

[0544] In embodiments, the therapy reverses or prevents excessive production of one or more inflammatory cytokines in a patient when administered. In embodiments, the inflammatory cytokine is one or more of IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

[0545] In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms associated with a coronavirus infection.

[0546] Coronaviruses (CoVs) are members of the family Coronaviridae, including betacoronavirus and alphacoronavirus—respiratory pathogens that have relatively recently become known to invade humans. The Coronaviridae family includes such betacoronavirus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43. Alphacoronavirus includes, e.g., HCoV-NL63 and HCoV-229E. In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms of infection with any of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43. Alphacoronavirus includes, e.g., HCoV-NL63 and HCoV-229E.

[0547] Without wishing to be bound by theory, coronaviruses invade cells through utilization of their “spike” surface glycoprotein that is responsible for viral recognition of Angiotensin Converting Enzyme 2 (ACE2), a transmembrane receptor on mammalian hosts that facilitate viral entrance into host cells. (Zhou et al., A pneumonia outbreak associated with anew coronavirus of probable bat origin, Nature 2020).

[0548] Symptoms associated with coronavirus infections include, but are not limited to, fever, tiredness, dry cough, aches and pains, shortness of breath and other breathing difficulties, diarrhea, upper respiratory symptoms (e.g., sneezing, runny nose, nasal congestion, cough, sore throat), and / or pneumonia. In embodiments, the present compositions and methods are useful in treating or mitigating any of these symptoms.

[0549] In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms of infection with SARS-CoV-2, including Coronavirus infection 2019 (COVID-19), caused by SARS-CoV-2 (e.g., 2019-nCoV).

[0550] In some settings, including subjects afflicted with coronavirus infections, it is possible that the morbidity and mortality of pulmonary viral infection is related to an exaggerated or overwhelming inflammatory response. In varying clinical circumstances this response can be described as “cytokine response syndrome”, “cytokine storm”, or “secondary hemophagocytic lymphohistiocytosis” (sHLH). In embodiments, the present compositions and methods are useful in treating or mitigating any of these exaggerated or overwhelming inflammatory responses. Collectively it is surmised that these highly proinflammatory states can lead to death due to pulmonary collapse such as acute respiratory distress syndrome (ARDS) or systemic, multi-organ failure affecting organs such as liver, kidney, heart and brain. In embodiments, the present cells treat or mitigate a “cytokine response syndrome”, “cytokine storm”, or “secondary hemophagocytic lymphohistiocytosis” (sHLH).

[0551] In embodiments, COVID-19 is characterized, in part, by elevation of Interleukin-2 (IL-2), Interleukin-7 (IL-7), granulocyte colony stimulating factor (GCSF), interferon-gamma inducible protein 10, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP1a), and tumor necrosis factor-alpha (TNFα). In embodiments, the present compositions and methods are useful in treating or mitigating increases of any of these factors.

[0552] In embodiments, the present cells prevent a COVID-19 patient from having a disease that develops from respiratory distress to cytokine storm.

[0553] In embodiments, the present cells treat or mitigate ARDS.

[0554] In some embodiments, a cytokine storm is associated with COVID-19 and is treated or mitigated via a method comprising administering to a subject in need thereof an effective amount of cells effective for the treatment of a coronavirus infection and / or a cytokine storm associated with a coronavirus infection, wherein the subject has abnormal (e.g. increased or decreased) expression or activity of one or more of IL-6, IL-1, TNF, interferon-γ, CXCL10, CCL7, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, CCL2 / MCP-1, CCL5 / RANTES, CCL7 / MCP-3, MCP-2, tumor necrosis factor-alpha (TNFα), interferon-γ (IFNγ), CXCL10, CXC3, Granulocyte colony stimulatory factor (GCSF), Macrophage inflammatory protein 1 alpha (MIP-1a), IL-22, and Interferon gamma induced protein 10 (IP-10).

[0555] In some embodiments, the subject has a modulated (e.g. decreased or increased) expression or activity of one or more of IL-6, IL-1, TNF, interferon-γ, CXCL10, CCL7, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, CCL2 / MCP-1, CCL5 / RANTES, CCL7 / MCP-3, MCP-2, tumor necrosis factor-alpha (TNFα), interferon-γ (IFNγ), CXCL10, CXC3, Granulocyte colony stimulatory factor (GCSF), Macrophage inflammatory protein 1 alpha (MIP-1a), IL-22, and Interferon gamma induced protein 10 (IP-10).

[0556] In embodiments, the disease / indication is associated with one or more cancers. The one or more cancers may comprise: adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann Syndrome, bile duct caner (Cholangiocarcinoma), Birt-Hogg Dube Syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, breast cancer (inflammatory), breast cancer (metastatic), breast cancer in men, carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma tumor, ependymoma, esophageal cancer, Ewing Sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor GIST, germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, leukemia—acute lymphoblastic—ALL, leukemia, acute lymphocytic—ALL, leukemia—acute myeloid—ALL, leukemia—acute myeloid—AML, leukemia—B-cell prolymphocytic leukemia and hairy cell leukemia, leukemia—chronic lymphocytic—CLL, leukemia—chronic myeloid—CML, leukemia—chronic t-cell lymphocytic, leukemia—eosinophilic, Li-Fraumeni Syndrome, liver cancer, lung cancer—non-small cell, lung cancer—small cell, lymphoma—Hodgkin, lymphoma—Non-Hodgkin, Lynch Syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH / MYH—associated polyposis, myelodysplastic syndromes—MDS, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers Syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma—Kaposi, sarcomas of specific organs, sarcomas—soft tissue, skin cancer (non-melanoma), skin cancer (melanoma), small bowel cancer, stomach cancer, testicular cancer, thyoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, unknown primary, uterine cancer, vaginal cancer, Von Hippel-Lindau Syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner Syndrome, Wilms tumor, and xeroderma pigmentosum.

[0557] In an aspect, the present disclosure provides a method for treating a cancer in a patient in need thereof. The method comprising administering to the cancer patient a therapeutically-effective amounts of any herein-disclosed cytotoxic lymphocyte.

[0558] An aspect of the present disclosure is a method for killing a cancer cell. The method comprising steps of: (1) obtaining a herein-disclosed cytotoxic lymphocyte and (2) contacting cytotoxic lymphocyte with the cancer. In some cases, the cancer cell is in vivo.

[0559] Yet another aspect of the present disclosure is a method for treating a cancer patient in need thereof. The method comprising a step of administering to the cancer patient a therapeutically-effective amounts of a herein-disclosed cytotoxic lymphocyte.

[0560] In aspects, the present disclosure provides a method of treating cancer, comprising: (a) obtaining an isolated cytotoxic lymphocyte comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene; and (b) administering the isolated cytotoxic lymphocyte to a subject in need thereof.

[0561] In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell.

[0562] In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0563] In embodiments, the cytotoxic lymphocyte is an NK cell.

[0564] In embodiments, the cancer is a blood cancer. In embodiments, the cancer is a solid tumor. In embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), and Meigs' syndrome.

[0565] The cytotoxic lymphocyte of the present disclosure may be administered systemically (e.g., via a vein or artery) or may be introduced into a tumor or in the vicinity of the tumor.Administration and Formulations

[0566] In some embodiments, the present disclosure relates to compositions described herein in the form of a pharmaceutical composition.

[0567] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0568] In embodiments, the first pharmaceutical composition comprises the population of isolated lymphoid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated myeloid lineage cells.

[0569] In some embodiments, the first pharmaceutical composition comprises the population of isolated myeloid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated lymphoid lineage cells. In some cases, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or sequentially. The first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the first pharmaceutical composition administered before the second pharmaceutical composition or the first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the second pharmaceutical composition administered before the first pharmaceutical composition.

[0570] In various embodiments, the first pharmaceutical composition comprises both the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells.

[0571] In various embodiments, the present invention pertains to pharmaceutical compositions comprising the recombinantly engineered cells described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the present invention pertains to pharmaceutical compositions comprising the iPSC-derived cells of the lymphoid lineage, including cytotoxic lymphocytes, iPSC-derived cells of the myeloid lineage, e.g., monocytes which can be differentiated into functional M1 and M2 macrophages having enhanced cytokine secretion and tumor cell-killing activity, and / or synthetic RNA molecules encoding the gene-editing protein or expression cassettes for expressing a protein of interest, e.g., a CAR or for expressing or overexpressing a cytokine.

[0572] Therapeutic treatments comprise the use of one or more routes of administration and of one or more formulations that are designed to achieve a therapeutic effect at an effective dose, while minimizing toxicity to the subject to which treatment is administered. Illustrative formulations / compositions of the present disclosure include engineered cells along with a suitable delivery reagent, e.g., a liquid carrier.

[0573] In various embodiments, the effective dose is an amount that substantially avoids cell toxicity in vivo. In various embodiments, the effective dose is an amount that substantially avoids an immune reaction in a human subject. For example, the immune reaction may be an immune response mediated by the innate immune system. Immune response can be monitored using markers known in the art (e.g., cytokines, interferons, TLRs). In some embodiments, the effective dose obviates the need for treatment of the human subject with immune suppressants agents (e.g., B18R) used to moderate the residual toxicity.

[0574] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective, as described herein. The formulations may easily be administered in a variety of dosage forms such as injectable solutions and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered, and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art. Pharmaceutical preparations may additionally comprise delivery reagents (a.k.a. “vehicles”, a.k.a. “delivery vehicles”) and / or excipients. Pharmaceutically acceptable delivery reagents, excipients, and methods of preparation and use thereof, including methods for preparing and administering pharmaceutical preparations to patients (a.k.a. “subjects”) are well known in the art, and are set forth in numerous publications, including, for example, in US Patent Appl. Pub. No. US 2008 / 0213377, the entirety of which is incorporated herein by reference.

[0575] The present pharmaceutical compositions can comprise excipients, including liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0576] In embodiments, the composition is formulated for one or more of intrathecal, intra-lesional, intra-coronary, intravenous (IV), intra-articular, intramuscular, and intra-endobronchial administration and administration via intrapancreatic endovascular injection, intra-nucleus pulposus, lumbar puncture, intra-myocardium, transendocardium, intra-fistula tract, intermedullary space, intradural space and leg injection.

[0577] In embodiments, the composition is formulated for infusion. In some embodiments, the composition is formulated for infusion, wherein the composition is delivered to the bloodstream of a subject or patient through a needle in a vein of the subject or patient through a peripheral line, a central line, a tunneled line, an implantable port, and / or a catheter. In some embodiments, the subject or patient may also receive supportive medications or treatments, such as hydration, by infusion. In some embodiments, the composition is formulated for intravenous infusion. In some embodiments, the infusion is continuous infusion, secondary intravenous therapy (IV), and / or IV push. In some embodiments, the infusion of the composition may be administered through the use of equipment selected from one or more of an infusion pump, hypodermic needle, drip chamber, peripheral cannula, and pressure bag.

[0578] In embodiments, the method of treating a subject comprises administering a cell of the present disclosure to a subject in need thereof. In embodiments, the cell is formulated for therapeutic use. In embodiments, the cell is suitable for administration to a human subject. In embodiments, the method is conducted in vivo.

[0579] In numerous embodiments, the administering is intravenous, intraarterial, intratumoral, or injected in the vicinity of a tumor.

[0580] In embodiments when a synthetic RNA molecule encoding a temperature-sensitive gene-editing protein is administered, the method comprises reducing the body temperature of a subject, optionally via whole-body hypothermia. In embodiments, the body temperature of the subject is reduced by from about 0.5° C. to about 1° C. In embodiments, the body temperature of the subject is reduced by from about 1° C. to about 1.5° C. In embodiments, the body temperature of the subject is reduced by from about 1.5° C. to about 2° C. In embodiments, the body temperature of the subject is reduced by from about 2° C. to about 2.5° C. In embodiments, the body temperature of the subject is reduced by from about 2.5° C. to about 3° C. In embodiments, the body temperature of the subject is reduced by from about 3° C. to about 3.5° C. In embodiments, the body temperature of the subject is reduced by from about 3.5° C. to about 4° C. In embodiments, the body temperature of the subject is reduced by from about 4° C. to about 4.5° C. In embodiments, the body temperature of the subject is reduced by from about 4.5° C. to about 5° C. In embodiments, the body temperature of the subject is reduced by from about 5° C. to about 5.5° C. In embodiments, the body temperature of the subject is reduced by from about 5.5° C. to about 6° C. In embodiments, the body temperature of the subject is reduced by from about 6° C. to about 6.5° C. In embodiments, the body temperature of the subject is reduced by from about 6.5° C. to about 7° C. In embodiments, the body temperature of the subject is reduced by from about 7° C. to about 7.5° C. In embodiments, the body temperature of the subject is reduced by from about 7.5° C. to about 8° C. In embodiments, the body temperature of the subject is reduced by from about 8° C. to about 8.5° C. In embodiments, the body temperature of the subject is reduced by from about 8.5° C. to about 9° C. In embodiments, the body temperature of the subject is reduced by from about 9° C. to about 9.5° C. In embodiments, the body temperature of the subject is reduced by from about 9.5° C. to about 10° C. In embodiments, the body temperature of the subject is reduced by from about 10° C. to about 10.5° C. In embodiments, the body temperature of the subject is reduced by from about 10.5° C. to about 11° C. In embodiments, the body temperature of the subject is reduced by from about 11° C. to about 11.5° C. In some embodiments, the reducing the body temperature of the subject is performed for a specific amount of time. In some embodiments, the specific amount of time is from about 15 minutes to about 30 minutes. In some embodiments, the specific amount of time is from about 30 minutes to about 45 minutes. In some embodiments, the specific amount of time is from about 45 minutes to about 60 minutes. In embodiments, the specific amount of time is from about 1 hour to about 1.5 hours. In embodiments, the specific amount of time is from about 1.5 hours to about 2 hours. In embodiments, the specific amount of time is from about 2 hours to about 2.5 hours. In embodiments, the specific amount of time is from about 2.5 hours to about 3 hours. In embodiments, the specific amount of time is from about 3 hours to about 3.5 hours. In embodiments, the specific amount of time is from about 3.5 hours to about 4 hours. In embodiments, the specific amount of time is from about 4 hours to about 4.5 hours. In embodiments, the specific amount of time is from about 4.5 hours to about 5 hours. In embodiments, the specific amount of time is from about 5 hours to about 5.5 hours. In embodiments, the specific amount of time is from about 5.5 hours to about 6 hours. In embodiments, the specific amount of time is from about 6 hours to about 6.5 hours.

[0581] In embodiments when a synthetic RNA molecule encoding a temperature-sensitive gene-editing protein is administered, the method comprises applying one or more cooling elements to a cell or tissue in vivo to reduce temperature, the cooling element optionally being a cryocompression device. In embodiments, the temperature is reduced by from about 0.5° C. to about 1° C. In embodiments, the temperature is reduced by from about 1° C. to about 1.5° C. In embodiments, the temperature is reduced by from about 1.5° C. to about 2° C. In embodiments, the temperature is reduced by from about 2° C. to about 2.5° C. In embodiments, the temperature is reduced by from about 2.5° C. to about 3° C. In embodiments, the temperature is reduced by from about 3° C. to about 3.5° C. In embodiments, the temperature is reduced by from about 3.5° C. to about 4° C. In embodiments, the temperature is reduced by from about 4° C. to about 4.5° C. In embodiments, the temperature is reduced by from about 4.5° C. to about 5° C. In embodiments, the temperature is reduced by from about 5° C. to about 5.5° C. In embodiments, the temperature is reduced by from about 5.5° C. to about 6° C. In embodiments, the temperature is reduced by from about 6° C. to about 6.5° C. In embodiments, the temperature is reduced by from about 6.5° C. to about 7° C. In embodiments, the temperature is reduced by from about 7° C. to about 7.5° C. In embodiments, the temperature is reduced by from about 7.5° C. to about 8° C. In embodiments, the temperature is reduced by from about 8° C. to about 8.5° C. In embodiments, the temperature is reduced by from about 8.5° C. to about 9° C. In embodiments, the temperature is reduced by from about 9° C. to about 9.5° C. In embodiments, the temperature is reduced by from about 9.5° C. to about 10° C. In embodiments, the temperature is reduced by from about 10° C. to about 10.5° C. In embodiments, the temperature is reduced by from about 10.5° C. to about 11° C. In embodiments, the temperature is reduced by from about 11° C. to about 11.5° C.

[0582] In embodiments when a synthetic RNA molecule encoding a temperature-sensitive gene-editing protein is administered, the applying one or more cooling elements to a cell or tissue in vivo to reduce temperature is performed for a specific amount of time. In some embodiments, the specific amount of time is from about 15 minutes to about 30 minutes. In some embodiments, the specific amount of time is from about 30 minutes to about 45 minutes. In some embodiments, the specific amount of time is from about 45 minutes to about 60 minutes. In some embodiments, the specific amount of time is from about 1 hour to about 1.5 hours. In some embodiments, the specific amount of time is from about 1.5 hours to about 2 hours. In some embodiments, the specific amount of time is from about 2 hours to about 2.5 hours. In some embodiments, the specific amount of time is from about 2.5 hours to about 3 hours. In some embodiments, the specific amount of time is from about 3 hours to about 3.5 hours. In some embodiments, the specific amount of time is from about 3.5 hours to about 4 hours. In some embodiments, the specific amount of time is from about 4 hours to about 4.5 hours. In some embodiments, the specific amount of time is from about 4.5 hours to about 5 hours. In some embodiments, the specific amount of time is from about 5 hours to about 5.5 hours. In some embodiments, the specific amount of time is from about 5.5 hours to about 6 hours. In some embodiments, the specific amount of time is from about 6 hours to about 6.5 hours.

[0583] Further description of temperature-sensitive cell administration is found in WO2021 / 231549. The entire contents of which are incorporated by reference in their entirety.

[0584] In some embodiments, the present invention relates to one or more administration techniques described in U.S. Pat. Nos. 5,711,964; 5,891,468; 6,316,260; 6,413,544; 6,770,291; and 7,390,780, the entire contents of which are hereby incorporated by reference in their entireties.Lipids Cell Contacting Transfection

[0585] In embodiments, the present invention relates delivery of the present synthetic RNA molecules via a lipid. In some embodiments, mRNAs encoding a gene-editing protein and / or a reprogramming factor are delivered via a lipid.

[0586] In embodiments, the lipid is a compound of Formula (I)wherein: Q1, Q2, Q3, and Q4 are independently an atom or group capable of adopting a positive charge;

[0588] A1 and A2 are independently null, H, or optionally substituted C1-C6 alkyl;

[0589] L1, L2, and L3 are independently null, a bond, (C1-C20)alkanediyl, (halo)(C1-C20)alkanediyl, (hydroxy)(C1-C20)alkanediyl, (alkoxy)(C1-C20)alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, or any combination of the aforementioned optionally linked by one or more of an ether, an ester, an anhydride, an amide, a carbamate, a secondary amine, a tertiary amine, a quaternary ammonium, a thioether, a urea, a carbonyl, or an imine;

[0590] R1, R2, R3, R4, R5, R6, R7, and R8 are independently null, H, (C1-C60)alkyl, (halo)(C1-C60)alkyl, (hydroxy)(C1-C60)alkyl, (alkoxy)(C1-C60)alkyl, (C2-C60)alkenyl, (halo)(C2-C60)alkenyl, (hydroxy)(C2-C60)alkenyl, (alkoxy)(C2-C60)alkenyl, (C2-C60)alkynyl, (halo)(C2-C60)alkynyl, (hydroxy)(C2-C60)alkynyl, (alkoxy)(C2-C60)alkynyl, wherein at least one of R1, R2, R3, R4, R5, R6, R7, and R8 comprises at least two unsaturated bonds; and x, y, and z are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0591] In embodiments, the lipid is a compound of Formula (II):wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, and R28 are independently H, halo, OH, (C1-C6)alkyl, (halo)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclo; and

[0593] i, j, k, m, s, and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0594] In embodiments, the lipid is a compound of Formula (III):wherein L4, L5, L6, and L7 are independently a bond, (C1-C20)alkanediyl, (halo)(C1-C20)alkanediyl, (hydroxy)(C1-C20)alkanediyl, (alkoxy)(C1-C20)alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, —(CH2)v1—C(O)—, —((CH2)v1—O)v2—, or —((CH2)v1—C(O)—O)v2—;

[0596] R29, R30, R31, R32, R33, R34, and R35 are independently H, (C1-C60)alkyl, (halo)(C1-C60)alkyl, (hydroxy)(C1-C60)alkyl, (alkoxy)(C1-C60)alkyl, (C2-C60)alkenyl, (halo)(C2-C60)alkenyl, (hydroxy)(C2-C60)alkenyl, (alkoxy)(C2-C60)alkenyl, (C2-C60)alkynyl, (halo)(C2-C60)alkynyl, (hydroxy)(C2-C60)alkynyl, (alkoxy)(C2-C60)alkynyl, wherein at least one of R29, R30, R31, R32, R33, R34, and R35 comprises at least two unsaturated bonds;

[0597] v, v1 and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0598] In embodiments, the lipid is a compound of Formula (IV):wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0600] In embodiments, the lipid is a compound of Formula (V):

[0601] In embodiments, the lipid is a compound of Formula (VI):

[0602] In embodiments, the lipid is a compound of Fo...

Examples

example 1

Cell Preparation Methods and Results

[1094]FIG. 1A shows schematic of cellular production methods used in this Example. mRNA-based cellular reprogramming (fibroblast to iPS cell) and gene-editing (beta-2-microglobulin (B2M) knockout) were employed. Further, edited cells were differentiated into cytotoxic lymphoid cells. FIG. 1B illustrates the differentiated cytotoxic lymphoid cells killing cancer cells.

[1095]Fibroblast cells were obtained from a human subject and reprogrammed to iPS cells using an mRNA-based reprogramming.

[1096]Efficient targeting of defined loci in iPSCs using messenger RNA (mRNA)-encoded gene-editing endonucleases comprising DNA-binding domains containing novel linker region was undertaken (e.g., a gene-editing protein comprising a DNA binding domain having at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (SEQ ID NO: 65; the “*RVD*” corresponds to the dinucleotide “xy” of SEQ ID NO: 22). Exon 3 of B2M, a key component of MHC class I molecules was tar...

example 2

Cells Characterization Methods and Results

[1114]Phenotypical and functional characterization assays were used to evaluate cells of Example 1. Phenotypical characterization used flow cytometry staining of surface markers, specifically CD56 / CD16 (e.g., gated on CD56). CD56 / NKG2D, CD56 / CD45, CD56 / CD3, CD56 / CD244, CD56 / CD94 / NKG2A, CD56 / NKp46, CD56 / NKp44, CD56 / KIRs, CD56 / TRAIL, and CD56 / FASL were also assessed (e.g., gated on CD56). See, FIG. 10A to FIG. 10C and the below tables:

[1115]Data in this first table characterize PBMC-Isolated NK Cells vs. iPSC-Derived NK Cells from suspension round 1:

WTB2M− / −IsolatedPopulationPopulationPopulationPopulationNK Cells1212% of25.6%74.4%32.9%67.1%PopulationCD5677.5%82.1%22.3%84.9%68.2%CD1616.3%2.06%0.25%8.23%21.3%CD328.0%76.1%20.0%61.5%22.9%CD4597.8%99.1%99.4%99.8%99.8%NKG2D9.17%20.9%59.3%17.0%43.8%

[1116]Data in this second table characterize iPSC-Derived NK Cells—AggreWell™ vs. iPSC-Derived NK Cells from suspension round 2

AggreWell ™SuspensionWTB2M−...

example 3

B2M-HLA-E Insertion

[1128]In this example, repair template (the B2M-HLA-E repair template) comprising the B2M coding sequence, and the HLA-E (Major Histocompatibility Complex, Class I, E) coding sequence was inserted into a beta-2-microglobulin (B2M) edit. Here, iPSCs having their B2M gene edited, as disclosed herein, are contacted with a repair template comprising the coding sequence for HLA-E. Alternately, un-edited iPSCs are contacted with the gene-editing components to edit the B2M gene along with a repair template comprising the coding sequence for HLA-E. In both cases, the resulting cell (either as in iPSC or when differentiated into a cytotoxic lymphocyte of the lymphoid or differentiated into cells of the myeloid lineage, e.g., macrophages, or mesenchymal stromal / stem cells, or hematopoietic stem cells) will have an edited B2M gene and will express, in its place, HLA-E.

[1129]As shown in FIG. 11A the B2M signal peptide sequence (B2M_sp), which is contained entirely within Exon...

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a National Stage Entry of International Application No. PCT / US2023 / 066464, which claims the benefit of U.S. Provisional Patent Application No. 63 / 337,166, filed May 1, 2022, and U.S. Provisional Patent Application No. 63 / 342,144, filed May 15, 2022, each of which is entirely incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on May 1, 2023, is named 61057-719_601.xml and is 81,727 bytes in size.BACKGROUND

[0003] Methods for reprogramming differentiated cells into pluripotent cells and methods for gene-editing cells have progressed greatly over recent years. However, there remain unmet needs for improved methods for reprogramming and gene editing cells.SUMMARY

[0004] Accordingly, the present disclosure provides improved methods for reprogramming and gene editing cells.

[0005] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0006] In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR), e.g., a CAR which comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In some cases, the antigen binding region binds to one or more tumor antigens. In various cases, the CAR comprises an antigen binding region that binds to ROR1. In embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine. In various embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein and a single-stranded or double-stranded repair template which encodes a chimeric antigen receptor (CAR). In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the CAR inserts into the break. In these embodiments, the cell in the subject expresses the CAR.

[0007] In numerous embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein and a single-stranded or double-stranded repair template which encodes a cytokine. In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the cytokine inserts into the break. In these embodiments, the cell in the subject expresses or over expresses the cytokine.

[0008] In various embodiments, the isolated lymphoid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes; wherein steps (5) and (6) occur in an adherent culturing vessel.

[0009] In embodiments, the isolated myeloid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a myeloid progenitor medium; and (6) culturing the cells of step (5) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5) and (6) occur in a bioreactor.

[0010] Another aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for treating a cancer.

[0011] Yet another aspect of the present disclosure is a method for killing a cancer cell or for inhibiting the proliferation of a cancer cell. The method comprising contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0012] In some embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vitro.

[0013] In various embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vivo.

[0014] Yet a further aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for killing a cancer cell or for inhibiting the proliferation of a cancer cell.

[0015] In an aspect, the present disclosure provides a method for manufacturing a plurality of population of cells comprising a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells for treating a cancer, for killing a cancer cell, and / or for inhibiting the proliferation of a cancer cell. The method a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor.

[0016] An aspect of the present disclosure is a method for manufacturing a population of cells that is enriched for cytotoxic lymphocytes. The method comprises steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes. In this aspect, steps (5) and (6) occur in an adherent culturing vessel. When CD34+ cells are selected, the embryoid bodies may be first chemically and / or mechanically dissociated.

[0017] Another aspect of the present disclosure is a method for killing a cancer cell. The method comprising steps of: (1) obtaining a herein-disclosed cytotoxic lymphocyte and (2) contacting cytotoxic lymphocyte with the cancer. In some cases, the cancer cell is in vivo.

[0018] Yet another aspect of the present disclosure is a method for treating a cancer patient in need thereof. The method comprising a step of administering to the cancer patient a therapeutically-effective amounts of a herein-disclosed cytotoxic lymphocyte.

[0019] In an aspect, the present disclosure provides a pharmaceutical composition comprising a herein-disclosed cytotoxic lymphocyte and a pharmaceutically acceptable carrier or excipient.

[0020] In another aspect, the present disclosure provides a composition comprising a cell comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, wherein the cell is a cytotoxic lymphocyte from a lymphoid lineage cell, e.g., an NK cell, or the cell is from a myeloid lineage, e.g., a macrophage, or the cell is a mesenchymal stromal / stem cell, or the cell is a hematopoietic stem cell.

[0021] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated NK cell of any herein-disclosed aspect or embodiment.

[0022] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated myeloid cell of any herein-disclosed aspect or embodiment, e.g., a macrophage.

[0023] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated mesenchymal stromal / stem cell of any herein-disclosed aspect or embodiment.

[0024] An aspect of the present disclosure is a method of making an engineered cell comprising a disruption in a beta-2-microglobulin (B2M) gene. The method comprising steps of (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins; and (c) differentiating the iPS cell into a differentiated cell. In this aspect, the differentiated cell is a cytotoxic lymphocyte from a lymphoid cell lineage or is from a myeloid cell lineage, e.g., a macrophage.

[0025] Another aspect of the present disclosure is a method of treating cancer. The method comprising steps of obtaining an isolated cell comprising a genetically engineered disruption in a B2M gene and administering the isolated cell to a subject in need thereof. In this aspect, the cytotoxic lymphocyte is a lymphoid cell or a CAR-myeloid cell or a CAR-mesenchymal stromal / stem cell.

[0026] Yet another aspect of the present disclosure is a composition comprising an isolated cytotoxic lymphocyte comprising a gene edit in a CD16a gene, wherein the cytotoxic lymphocyte is a lymphoid lineage cell, e.g., an NK cell.

[0027] An aspect of the present disclosure is a method for producing macrophages from an induced a pluripotent stem cell (iPSC). The method comprises steps of: (1) obtaining an iPSC; (2) culturing the iPSC in a first medium for about three days; (3) culturing the iPSC in a second for about four days; (4) culturing the iPSC in a monocyte differentiating medium for at least seven days, thereby obtaining monocytes (5) isolating the monocytes; (6) culturing the monocytes for about four days; (7) culturing the monocytes in the presence of M-CSF for three to four days, thereby obtaining macrophages; and (8) harvesting the macrophages. In some embodiments, the macrophages are further contacted with interferon gamma (IFN-γ) and / or lipopolysaccharide (LPS) to obtain M1 macrophages and / or the macrophages are further contacted with IL-4 to obtain M2 macrophages.

[0028] In embodiments, the macrophages, e.g., the M1 and M2 macrophages, are capable of killing cancer cells.

[0029] In embodiments, the iPSC was reprogrammed from a differentiated or non-pluripotent cell.

[0030] In embodiments, the iPSC or a progenitor cell was gene-edited. In some cases, the iPSC or the progenitor cell was gene-edited to knockout the beta-2 microglobulin (B2M) gene.

[0031] In embodiments, the gene-editing comprises transfection of a repair template.

[0032] In embodiments, the repair template includes the coding sequence for one or more of HLA class I histocompatibility antigen, alpha chains (HLAs).

[0033] In embodiments, the repair template comprises a TTAGGG motif for decreasing synthetic oligodeoxynucleotides (ODNs)-related activation of pro-inflammatory responses and / or the cell is transfected with a TTAGGG motif separate from the repair template.

[0034] In embodiments, the differentiated or non-pluripotent cell was contacted with resveratrol before reprogramming.

[0035] In embodiments, the iPSC was contacted with resveratrol before gene-editing and / or the iPSC was contacted with resveratrol after gene-editing.

[0036] Another aspect is an isolated macrophage obtained by a herein disclosed method.

[0037] Yet another aspect is pharmaceutical composition comprising a herein disclosed isolated macrophage of and a pharmaceutically-acceptable excipient.

[0038] In an aspect, the disclosure provides an isolated M1 macrophage and / or an isolated M2 macrophage obtained by a herein disclosed method.

[0039] In yet another aspect, the disclosure provides a pharmaceutical composition comprising a herein disclosed isolated M1 macrophage and / or an isolated M2 macrophage and a pharmaceutically-acceptable excipient.

[0040] In another aspect, the disclosure provides method for treating a cancer comprising in vivo administering to a subject in need a herein disclosed pharmaceutical composition.

[0041] An aspect of the present disclosure is a method for decreasing synthetic oligodeoxynucleotides (ODNs)-related activation of pro-inflammatory responses, the method comprising transfecting a cell with an ODN comprising a TTAGGG motif.

[0042] In embodiments, the ODN is a double stranded ODN (dsODN) and comprise a repair template.

[0043] In embodiments, the TTAGGG motif is attached to the 5′ and / or the 3′ end of the repair template.

[0044] In embodiments, the ODN is a single stranded ODN (ssODN) and does not comprise a repair template.

[0045] In embodiments, the cell is transfected with a synthetic nucleic acid encoding a gene-editing protein along with a repair template.

[0046] Another aspect is an isolated cell obtained by a method relating to an ODN comprising a TTAGGG motif.

[0047] Yet another aspect is a method for enhancing the efficiency of gene-editing, the method comprising contacting a cell with resveratrol before gene-editing.

[0048] In embodiments, contacting the cell with resveratrol arrests the cell in S / G2 phase.

[0049] In embodiments, the cell is further contacted with resveratrol after gene-editing.

[0050] In embodiments, gene-editing comprise transfection of a synthetic nucleic acid encoding a gene-editing protein.

[0051] In an aspect, the present disclosure provides a method for enhancing the efficiency of gene-editing, the method comprising contacting a cell that has been gene-edited with resveratrol.

[0052] In embodiments, the gene-editing comprise transfection of a synthetic nucleic acid encoding a gene-editing protein.

[0053] Any aspect or embodiment herein may be combined with any other aspect or embodiment as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1A shows a non-limiting schematic of the mRNA-based reprogramming and gene-editing, followed by differentiation of the present disclosure. FIG. 1B illustrates differentiated cells killing cancer cells.

[0055] FIG. 2 shows the design of the gene-editing scheme for beta-2-microglobulin (B2M); shown are the following sequences: TCATCCATCCGACATTGA (SEQ ID NO: 1), AGTTGACTTACTGAAG (SEQ ID NO: 2), AATGGAGAGAGAATTGAA (SEQ ID NO: 3).

[0056] FIG. 3 shows an RNA gel demonstrating gene-editing of B2M.

[0057] FIG. 4 shows a sequencing experiment that shows the 14 base pair deletion from a gene-edited B2M; shown are the following sequences from bottom to top: ACATTGAAGAATGGAG (SEQ ID NO: 4), ACATTGAAGTTGACTTACTGAAGAATGGAG (SEQ ID NO: 5), and TGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAAT GGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGT (SEQ ID NO: 6).

[0058] FIG. 5 shows RNA levels of B2M with or without IFN gamma activation (“IFNY”; two left bars are the B2M knockout, and the two right bars are naïve cells).

[0059] FIG. 6 shows a sequencing experiment that demonstrates heterozygosity of CD16a (at G147D dbSNP:rs443082, Y158H dbSNP:rs396716, and F176V dbSNP:rs396991); shown are the following sequences from top to bottom: GKGRKYFHHNSDFHIPKATLKDS (SEQ ID NO: 7), GKDRKYFHHNSDFYIPKATLKDS (SEQ ID NO: 8), KDSGSYFCRGLFGSKNVSSETVN (SEQ ID NO: 9), and KDSGSYFCRGLVGSKNVSSETVN (SEQ ID NO: 10).

[0060] FIG. 7A-7B shows images of control (PMBC-isolated) NK cells in co-culture with K-562 tumor cells, demonstrating NK Cell cytotoxicity of tumor cell (note immunothrombosis or “clumping”).

[0061] FIG. 8A-8B shows images of the gene edited and differentiated cells of the present disclosure (e.g., B2M knockout NK cells) in co-culture with K-562 tumor cells, demonstrating NK Cell cytotoxicity of tumor cell (note immunothrombosis or “clumping”).

[0062] FIG. 9A-FIG. 9H show results of the cytokine release assay with the Luminex MAGPIX. Unless indicated (i.e., “+IL2, IL15”), conditions are without added IL-2 or IL-15. Further, ratio of cells is indicated (1:1 or 3:1). As elsewhere herein, PBMC-NK are control NK cells. FIG. 9A shows interferon gamma. FIG. 9B shows IL-2. FIG. 9C shows IL-7. FIG. 9D shows IL-13. FIG. 9E shows MIP-1a. FIG. 9F shows MIP-1b. FIG. 9G shows TNFα. FIG. 9H shows GM-CSF.

[0063] FIG. 10A-FIG. 10D show flow cytometry data for a gene edited and differentiated cells of the present disclosure (e.g., B2M knockout NK cells) as described in the Examples.

[0064] FIG. 11A shows the structure for the B2M-HLA-E repair template. FIG. 11B shows an ideal target site for the B2M-HLA-A repair template is shown (SEQ ID NO: 11: MSRSVALAVLALLSLSGLEAIQ; and SEQ ID NO: 12 ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGC TATCCAGCgtgagtctctcctaccctcccgctc). FIG. 11C shows additional target binding sites (SEQ ID NO: 11 and SEQ ID NO: 12 are again shown). FIG. 11D shows a gel with sizes of two lines having the B2M-HLA-E repair template inserted. FIG. 11E includes graphs showing the intensities of signal and ratios thereof from the bands shown in FIG. 11D. FIG. 11F shows a gel with sizes of two lines having the B2M-HLA-E repair template inserted. FIG. 11G includes graphs showing the intensities of signal and ratios thereof from the bands shown in FIG. 11F. FIG. 11H shows relevant sequences in the B2M-HLA-E repair template.

[0065] FIG. 12A and FIG. 12B, show target site sequences and repair templates for replacing the phenylalanine (F) at position 158 of CD16a with a valine (V). Relevant sequences are shown in these figures.

[0066] FIG. 13 is a graphical representation of different protocols in the differentiation of cytotoxic lymphocytes.

[0067] FIG. 14 is an illustrative flow cytometry fluorescence map used in data analysis of cytotoxicity assays.

[0068] FIG. 15 are graphs showing percentages of cancer killed in 24 hours. The left data for each graph are cells that were not activated and the right data for each graph are cells that were activated with IL-15 and IL-2.

[0069] FIG. 16 are graphs showing the ability of cytotoxic lymphocytes to kill K692 cancer cells and their inability to kill NK-resistant cancer cells.

[0070] FIG. 17 is a scatter plot showing two distinct populations of cells.

[0071] FIG. 18 are scatter plots for cells derived from Protocol 2, 3, or 4 as illustrated in FIG. 13.

[0072] FIG. 19 is a cartoon showing methods for manufacturing mixed population iPS-cell derived lymphoid lineage cells and myeloid lineage cells for enhanced tumor cell killing.

[0073] FIG. 20 shows an overview of scalable iPSC differentiation into lymphoid and myeloid cells.

[0074] FIG. 21 includes photomicrographs showing morphology of iPSC to macrophage progenitor cells in the T75 and Bioreactor.

[0075] FIG. 22 includes graphs showing the sum of the viable macrophage progenitor cells harvested from the T75 (left) and bioreactor (right) throughout the culture period. * Indicates when a large harvest (>50% of total cells) was performed.

[0076] FIG. 23 is a graph assessing baseline macrophage cytotoxicity.

[0077] FIG. 24 includes florescent photomicrographs of mRNA transfection of macrophages.

[0078] FIG. 25 includes photomicrographs showing morphology of NK cells on the final day of the differentiation protocol (left) versus 24 hours post thaw (right).

[0079] FIG. 26 is a graph showing the effects of cryopreservation on the cytotoxicity of NK cells.

[0080] FIG. 27 includes a graph (left) and photomicrographs (right) of data from an isolated huPBMC mixed cell type cytotoxicity assay.

[0081] FIG. 28 includes data from cytotoxicity assay cytokine release heat maps with iPS derived macrophages and NK cells. * Indicates below level of detection. ** Indicates sample read N / A.

[0082] FIG. 29 includes photomicrographs showing iPSC derived immune cell clustering during a cytotoxicity assay after 24 hours.

[0083] FIG. 30 is a cartoon showing methods for rapid prototyping of macrophage gene-editing strategies for cancer immunotherapies.

[0084] FIG. 31 is a timeline showing efficient differentiation of iPSCs into macrophages.

[0085] FIG. 32 is a photomicrograph showing iPSC-macrophages displaying macrophage-like morphology following differentiation.

[0086] FIG. 33 is a schematic of an mRNA encoding a ROR1-CAR.

[0087] FIG. 34A includes florescent photomicrographs and FIG. 34B includes graphs of data from GFP-encoding mRNA transfected into macrophages.

[0088] FIG. 35A includes florescent photomicrographs and FIG. 35B includes graphs of data from macrophages transfected with mRNA encoding an ROR1-CAR.

[0089] FIG. 36 includes photomicrographs of data from a Zymosan bead phagocytosis assay.

[0090] FIG. 37 includes photomicrographs of data from a CD3ζ phosphorylation assay.

[0091] FIG. 38 includes photomicrographs (left) and a graph (right) of data from an SKOV3 cytotoxicity assay.

[0092] FIG. 39 is a cartoon showing a protocol for insertion of transgene.

[0093] FIG. 40 is a schematic of the structure of a ROR1-CAR transgene.

[0094] FIG. 41 is a gel showing insertion of a ROR1-CAR transgene into iPSC lines.

[0095] FIG. 42 is a cartoon showing methods for reprogramming fibroblasts into induced pluripotent stem cells (iPSCs), which are then differentiated into monocytes which are further differentiated into cancer killing macrophages.

[0096] FIG. 43A to FIG. 43C show general steps for the process of differentiating iPSCs to macrophage.

[0097] FIG. 43A shows iPSC to monocyte differentiation, FIG. 43B shows CD14+ magnetic bead positive selection, and FIG. 43C shows monocyte to macrophage differentiation.

[0098] FIG. 44 shows progressing of cells from an iPSC colony (top left), on day 3 mesoderm (top right), on day 7 hematopoietic stem cell (bottom left), and on day 14 monocyte (bottom right).

[0099] FIG. 45A to FIG. 45C are flow cytograms showing peaks marked by CD14 (FIG. 45A), CD45 (FIG. 45B), and CD163 (FIG. 45C) for cryopreserved PBMC-monocytes and for cryopreserved iPSC-monocytes flowed directly after thawing.

[0100] FIG. 46 shows monocyte cultures at day zero (left image) and at day 4 and upon activation with M-CSF (right image).

[0101] FIG. 47 shows macrophage cultures at day zero (left image) and at day 3 after (right image).

[0102] FIG. 48 is a graph showing ELISA on iPSC-Macrophage supernatants (1M cells / mL).

[0103] FIG. 49 shows the process for testing the cancer-cell killing ability of iPSC-macrophages of the present disclosure.

[0104] FIG. 50 is a flow cytometry scatter plot showing iPSC-derived macrophages killing of U2OS cancer cells in vitro.

[0105] FIG. 51 is a chart showing that iPSC-derived macrophages killed 45% of U2OS cancer cells in vitro.

[0106] FIG. 52 is a flow cytometry scatter plot showing iPSC-derived macrophages killing of MA011sk cells or donor fibroblasts in vitro.

[0107] FIG. 53 is a chart showing that iPSC-derived macrophages do not kill MA011sk cells or donor fibroblasts in vitro.

[0108] FIG. 54 is a cartoon showing methods for reducing an immune response by including a TTAGGG motif in an dsODN.

[0109] FIG. 55A to FIG. 55C shows that iMSC electroporated with a synthetic nucleic acid encoding a gene-editing protein and with a repair template comprising the code for GFP, expressed GFP 24 hours after electroporation (FIG. 55A), 72 hours after electroporation (FIG. 55B), and 28 days hours after electroporation and by passage 4 (FIG. 55C).

[0110] FIG. 56 is a blot showing results from gene-editing iMSCs with the gene-editing protein alone (lane 3) or with the gene-editing protein, the A151 oligo, dsODN repair template, and U11 (lane 4).

[0111] FIG. 57 are flow cytometry scatter plots for iMSCs that were gene edited along with the A151 ODN.

[0112] FIG. 58 is a cartoon showing the process of contacting Resveratrol with a cell in advance of gene editing.

[0113] FIG. 59 are graphs showing numbers of cells in S / G2 that were not pretreated with Resveratrol (left graph) and numbers of cells in S / G2 that were pretreated with Resveratrol (right graph).

[0114] FIG. 60 is a gel showing that Resveratrol pretreated fibroblasts have increased gene-editing efficiency.

[0115] FIG. 61 is a gel showing that that Resveratrol treatment after electroporation with gene-editing nucleic acids increased 1 kb insertion 1.6-fold in iPSC.

[0116] FIG. 62 is a gel showing that NU7441 (a DNA-PKs inhibitor that is known to inhibit NHEJ mediated DNA repair pathway) have increased gene-editing efficiency.

[0117] FIG. 63 is a gel showing that a maximum 32% of 1 kb insertion rate was observed using ssDNA repair template in iPSCs.DETAILED DESCRIPTIONMethods for Manufacturing Cytotoxic Lymphocytes

[0118] An aspect of the present disclosure is a method for manufacturing a population of cells that is enriched for cytotoxic lymphocytes. The method comprises steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes. In this aspect, steps (5) and (6) occur in an adherent culturing vessel. When CD34+ cells are selected, the embryoid bodies may be first chemically and / or mechanically dissociated.

[0119] In embodiments, the stem cell is an induced pluripotent stem (iPSC).

[0120] In some embodiments, the stem cell has a wild-type genome or has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene. In some cases, the stem cell has a biallelic disruption in a B2M gene.

[0121] In some cases, mRNA-reprogrammed iPSC lines with a biallelic knockouts of the beta-2 microglobulin (B2M) gene, a key component of MHC class I molecules, are obtained using an mRNA-encoded chromatin context-sensitive gene-editing endonuclease. The B2M-knockout iPSCs may be differentiated using a novel, fully suspension process that replaces specialized micropatterned culture vessels with a spheroid culture step. Additional details regarding B2M knockout iPSCs useful in the present disclosure are described in PCT / US2022 / 019020, the contents of which are incorporated herein by reference in its entirety.

[0122] In various embodiments, the bioreactor is suited for culturing shear-sensitive cells and / or does not require use of anti-foaming agents or shear protectants, e.g., a vertical wheel bioreactor such as a PBS Biotech vertical-wheel bioreactor.

[0123] In embodiments, the medium in step (2) is serum-free and feeder-free culture medium, e.g., an mTeSR™ medium.

[0124] In some embodiments, the medium in step (6) is a serum-free and feeder-free culture medium, e.g., a StemDiff™ NK medium.

[0125] In various embodiments, the adherent culturing vessel is a multi-well plate or a cell culturing flask.

[0126] In embodiments, the method provides from about 10-fold to about 100-fold more cytotoxic lymphocytes than obtained by a method in which each of the culturing steps comprise adherent culturing vessels; obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels; and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0127] In some embodiments, the cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells.

[0128] In various embodiments, the method provides from about 5-fold to about 30-fold more CD16+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0129] In embodiments, the method provides from about 5-fold to about 25-fold more NDG2D+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0130] In some embodiments, the method provides from about 2-fold to about 30-fold more NKp44+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0131] In various embodiments, the method provides from about 2-fold to about 8-fold more CD94+ cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels and / or obtained by a method in which steps (5) and (6) occur in bioreactor.

[0132] In embodiments, the method provides from about 2-fold more NKp46 cells than obtained by a method in which step (2) comprises a spheroid suspension culture and steps (3), (5), and (6) occur in adherent culturing vessels.

[0133] In some embodiments, the cytotoxic lymphocyte targets and kills cancer cells, e.g., a K562 cancer cell. In various embodiments, the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. In embodiments, the cytotoxic lymphocyte targets and kills at least 70% of cancer cells in a population within about 4 hours. In some embodiments, the cytotoxic lymphocyte targets and kills at least 80% of cancer cells in a population within about 24 hours.

[0134] In various embodiments, the cytotoxic lymphocyte has reduced cytotoxicity to an NK-resistant cancer cell, e.g., a NAMALWA cell.

[0135] In embodiments, the cytotoxic lymphocyte is a Natural Killer (NK) cell. In some cases, the NK cell is a mature NK cell.

[0136] In some embodiments, the cytotoxic lymphocyte is a Natural killer T (NKT) cell.

[0137] In various embodiments, the cytotoxic lymphocyte is a delta-gamma T cell.

[0138] In embodiments, the iPSC was reprogrammed from a somatic cell comprising contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors.Cytotoxic Lymphocytes

[0139] In embodiments, the present cytotoxic lymphocyte is of the lymphoid cell lineage or the myeloid cell lineage.

[0140] In some cases, the lymphoid cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell.

[0141] In some cases, the lymphoid cell is an NK cell, e.g., an NK-T cell. The NK cell may be a human cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0142] In various embodiment, the cytotoxic lymphocyte is reprogrammed from a stem cell, e.g., an iPSC, and differentiated into the cytotoxic lymphocyte.

[0143] In embodiments, the cytotoxic lymphocyte has a disruption in its beta-2-microglobulin (B2M) gene. In embodiments, the cytotoxic lymphocyte has a disruption in its beta-2-microglobulin (B2M) gene and expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0144] In embodiments, the cytotoxic lymphocyte is gene edited to express a high affinity variant of CD16a (See, FIG. 12A and FIG. 12B).

[0145] In embodiments, the myeloid lineage cell is a cell derived from, or derivable from, a common myeloid progenitor cell. In embodiments, the myeloid cell is a megakaryocyte, erythrocyte, mast cell, or myeloblast. In embodiments, the myeloid cell is a cell derived from, or derivable from, a myeloblast. In embodiments, the myeloid cell is a basophil, neutrophil, eosinophil, or monocyte. In embodiments, the myeloid cell is a cell derived from, or derivable from a monocyte. In embodiments, the myeloid cell is a macrophage. In embodiments, the myeloid cell is a dendritic cell.

[0146] In embodiments, the cytotoxic lymphocyte is an NK cell. In embodiments, the NK cell is a human cell. In embodiments, the NK cell is derived from somatic cell of a subject. In embodiments, the NK cell is derived from allogeneic or autologous cells. In embodiments, the NK cell is derived from an induced pluripotent stem (iPS) cell. In embodiments, the iPS is derived from reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors, optionally selected from Oct4, Sox2, cMyc, and Klf4. In embodiments, the iPS cell is derived from allogeneic or autologous cells. In embodiments, the NK cell expresses one or more of CD56 and CD16.

[0147] In embodiments, the NK cell expresses CD16a, which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V (See, FIG. 12A and FIG. 12B).

[0148] In embodiments, the NK cell does not express CD3.

[0149] In embodiments, the NK cell is CD56bright CD16dim / −. In embodiments, the NK cell is CD56dim CD16+. In embodiments, the NK cell is a NKtolerant cell, optionally comprising CD56bright NK cells or CD27− CD11b− NK cells. In embodiments, the NK cell is a NKcytotoxic, optionally comprising CD56dim NK cells or CD11b+ CD27− NK cells. In embodiments, the NK cell is a NKregulatory, optionally comprising CD56bright NK cells or CD27+ NK cells. In embodiments, the NK cell is a natural killer T (NKT) cell. In embodiments, the NK cell secretes one or more cytokines selected from interferon-gamma (IFN-g), tumor necrosis factor-alpha (TNF-a), tumor necrosis factor-beta (TNF-b), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b).

[0150] In embodiments, the present cytotoxic lymphocyte has reduced or eliminated cytotoxic lymphocyte fratricide, e.g., NK-cell fratricide. For instance, in embodiments, the present engineered NK cells surprisingly do not engage in NK cytotoxicity and therefore are able to survive despite disruptions, e.g., in beta-2-microglobulin (B2M).

[0151] In embodiments, the present cytotoxic lymphocyte is capable of self-activating. In embodiments, the present cytotoxic lymphocyte is capable of activating without the need for extracellular signals (e.g., cytokines), including signals that may be provided exogenously. In embodiments, the present cytotoxic lymphocyte does not require ex vivo stimulation for activity. In embodiments, the present cytotoxic lymphocyte is capable of self-activating in the absence of an interleukin, optionally selected from IL-2 and IL-15.

[0152] In embodiments, the present cytotoxic lymphocyte is capable of inducing tumor cell cytotoxicity. In embodiments, the present cytotoxic lymphocyte is capable of inducing tumor cell cytotoxicity in the absence of an interleukin, optionally selected from IL-2 and IL-15. Assays for assessing tumor cell cytotoxicity include in vivo anti-cancer response evaluation, as well as microscopic evaluation, e.g., a calcein acetoxymethyl (AM) staining-based microscopic method (See EXAMPLES and Chava et al. J Vis Exp. 2020 Feb. 22; (156): 10.3791 / 60714, the entire contents of which are incorporated by reference). Further, a colorimetric lactic dehydrogenase (LDH) measurement-based NK cell-mediated cytotoxicity assay may be employed (see Chava et al. J Vis Exp. 2020 Feb. 22; (156): 10.3791 / 60714, the entire contents of which are incorporated by reference).Scalable, Mixed Population iPS-Cell Derived Cytotoxic Lymphocytes and Myeloid Cells

[0153] Induced pluripotent stem cell (iPSC) therapies have the potential to treat a wide variety of devastating diseases. iPSC-derived lymphocytes (e.g., T cells and NK cells) engineered to express targeting molecules such as chimeric antigen receptors (CARs) have shown clinical promise to treat hematological malignancies. More recently, iPS cell-derived myeloid cells are being developed to treat both hematological malignancies and solid tumors due to the ability of these cells to infiltrate and modulate the tumor microenvironment. Despite preliminary success, several challenges still remain, including poor infiltration of cytotoxic lymphocytes into solid tumors and insufficient cytotoxicity of myeloid cells.

[0154] As is known in the art, an animal's immune system comprises a wide variety of immune cell types capable of contributing to an anti-cancer effect. And, in vivo, one type of immune cell promotes the cancer-killing ability of a second type of immune cell. Notably, NK cells are expert in killing cancer cells but rarely not infiltrate solid tumors alone and require recruitment by macrophages which have already infiltrated the solid tumor and, on the other hand, macrophages are less adept at killing cancer cells but expert in infiltrating solid tumors and secreting cytokines that recruit cancer killing cells. Thus, each type of immune cell has its function which work in cooperation with the other cell types to attack and kill cancer cells. Nonetheless, many cell-based cancer therapeutics in clinical trials employ one type of immune cell rather than a plurality of immune cell types as existent in vivo. Without wishing to be bound by theory, a multi-cell-type therapy comprising both lymphocyte and myeloid cells may work synergistically, enhancing cytotoxicity and efficacy.

[0155] This disclosure, e.g., in Examples 8 and 9, describes a scalable bioreactor-based process for parallel differentiation of mRNA reprogrammed iPSC into both CD14+ (>95% positive) macrophages and CD56bright / CD16dim NK cells. This process yielded 1×106 myeloid cells / ml and 3×105 lymphoid cells / ml, and is amenable to scaling to clinically relevant doses. In vitro, the lymphoid and myeloid cells showed synergistic tumor cell killing of SKOV3 ovarian cancer cells (combined: 15.6%; macrophage alone=2.2% (p<0.01); NK alone=7.5% (p<0.05); E:T=5:1). The combined cells showed increased expression of TNFα and demonstrated enhanced clustering and tumor cell engagement. To further improve the macrophages' ability to target and infiltrate solid tumors, macrophages were transfected with mRNA encoding a humanized ROR1-CAR protein. mRNA transfection increased cytotoxicity towards SKOV3 cells by 6-fold.

[0156] In conclusion, the present disclosure provides a scalable platform for generating iPSC-derived multi-cell-type therapies comprising both lymphoid and myeloid cells. These cells act synergistically to kill tumor cells in vitro. And, by closely mimicking natural cellular immunity, multi-cell-type cell therapies represent a new class of cell therapies that may play an important role in the development of new medicines for treating cancer.

[0157] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0158] In embodiments, the first pharmaceutical composition comprises the population of isolated lymphoid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated myeloid lineage cells.

[0159] In some embodiments, the first pharmaceutical composition comprises the population of isolated myeloid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated lymphoid lineage cells. In some cases, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or sequentially. The first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the first pharmaceutical composition administered before the second pharmaceutical composition or the first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the second pharmaceutical composition administered before the first pharmaceutical composition.

[0160] In various embodiments, the first pharmaceutical composition comprises both the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells.

[0161] In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR), e.g., a CAR which comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In some cases, the antigen binding region binds to one or more tumor antigens. In various cases, the CAR comprises an antigen binding region that binds to ROR1. In embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine. In some embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, optionally, wherein the cells express a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0162] In various embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a chimeric antigen receptor (CAR). In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the CAR inserts into the break. In these embodiments, the cell in the subject expresses the CAR.

[0163] In numerous embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a cytokine. In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the cytokine inserts into the break. In these embodiments, the cell in the subject expresses or over expresses the cytokine.

[0164] When the synthetic mRNA and / or the repair template is administered to a subject, the synthetic mRNA and / or the repair is combined with a lipid system comprising a compound of Formula (IV).

[0165] In various cases, transfection of a cell with synthetic nucleic acids for gene-editing may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In embodiments, the cell in the subject (e.g., which expresses the CAR and / or cytokine) is of the lymphoid lineage or is of the myeloid lineage.

[0167] In some embodiments, the isolated lymphoid lineage cell and / or the isolated myeloid lineage cell is derived from an induced pluripotent stem cell (iPSC). In some cases, the isolated lymphoid lineage cell and the isolated myeloid lineage cell is derived from the same iPSC. The iPSC comprises a genomic modification that expresses a chimeric antigen receptor (CAR) and / or the iPSC comprises a genomic modification that expresses or over expresses a cytokine. In various cases, the iPSC comprises a genomic modification which disrupts the beta-2-microglobulin (B2M) gene.

[0168] In various embodiments, the isolated lymphoid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes; wherein steps (5) and (6) occur in an adherent culturing vessel.

[0169] In numerous embodiments, the isolated lymphoid lineage cells comprise cytotoxic lymphocytes. In some cases, the isolated lymphoid lineage cells comprising cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells. In these embodiments, the cytotoxic lymphocyte targets and kills cancer cells and, in some cases, the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. The cytotoxic lymphocyte has reduced cytotoxicity to an NK-resistant cancer cell. In various cases, the cytotoxic lymphocyte is a Natural Killer (NK) cell, e.g., a mature NK cell, or is a cytotoxic T cell. In these embodiments, the cytotoxic lymphocyte is a Natural killer T (NKT) cell. In some cases, the NK cell expresses CD16a and / or the NK cell does not express CD3, and / or the NK cell is CD56bright CD16dim / −. In many cases, the NK cell secretes one or more cytokines selected from interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b). The cytotoxic lymphocyte may be a delta-gamma T cell. In numerous cases, the cytotoxic lymphocyte is further engineered to express a chimeric antigen receptor (CAR) and / or is further engineered to express or overexpress a cytokine.

[0170] In embodiments, the isolated myeloid lineage cells are manufactured by a method comprising steps of (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a myeloid progenitor medium; and (6) culturing the cells of step (5) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5) and (6) occur in a bioreactor.

[0171] In some embodiments, the isolated myeloid lineage cells comprise a megakaryocyte, erythrocyte, mast cell, myeloblast, dendritic cell, basophil, neutrophil, eosinophil, monocyte, or macrophage.

[0172] In various embodiments, the isolated myeloid lineage cells express one or more of CD11b, CD13, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., in amounts that are similar to amounts expressed by PBMC-derived cells.

[0173] In numerous embodiments, the isolated myeloid lineage cells have increased expression of CD80 and / or CD206, which is indicative of an activated state.

[0174] In embodiments, the isolated myeloid lineage cell is a macrophage. In some cases, the macrophage expresses one or more of CD11b, CD68, CD80, CD86, CD163, CD206, and SIRPα in amounts that are similar to amounts expressed by PBMC-derived cells and / or secretes one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells. In various cases, the macrophage expresses one or more of CD34, CD44, CD45, CD73, and CD90. In these embodiments, the method further comprises a step of differentiating the macrophages into M1 and / or M2 macrophages, e.g., by exposure to MCSF. And, the method may further comprise a step of polarizing the M1 macrophages with interferon gamma (IFN-γ) and / or lipopolysaccharide (LPS) and / or treating the M2 macrophages with IL-4. In these cases, the macrophages comprise M1 macrophages and / or M2 macrophages. The M1 macrophages and / or M2 macrophages secrete one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells.

[0175] In some embodiments, the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0176] In various embodiments, the isolated myeloid lineage cell is further engineered to express a chimeric antigen receptor (CAR).

[0177] In numerous embodiments, the isolated myeloid lineage cell is further engineered to express or overexpress a cytokine. In cases when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated.

[0178] In embodiments, the stem cell is an induced pluripotent stem (iPSC). In some cases, the stem cell stem has a wild-type genome or has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a biallelic disruption in a B2M gene. In various cases, the stem cell expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0179] In some embodiments, the iPSC was reprogrammed from a somatic cell and the method further comprises contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors. The one or more reprogramming factors may be selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In some cases, the somatic cell is selected from fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0180] In embodiments where the stem cell is an iPSC, the iPSC is further engineered to express a chimeric antigen receptor (CAR) and / or the iPSC is further engineered to express or overexpress a cytokine. In various embodiments, the isolated lymphoid lineage cells and the isolated myeloid lineage cells are manufactured by a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor. In cases when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated. In various cases, the stem cell is an induced pluripotent stem (iPSC).

[0181] In embodiments, the method of manufacturing provides at least 1×106 myeloid lineage cells / ml and at least 3×105 lymphoid lineage cells / ml.

[0182] In some embodiments, the method of manufacturing provides both CD14+ (>95% positive) macrophages and CD56bright / CD16dim NK cells.

[0183] In various embodiments, the method of manufacturing is amenable to scaling to clinically relevant doses.

[0184] In embodiments, the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells act synergistically to kill cancer cells.

[0185] In numerous embodiments, the administering is intravenous, intraarterial, intratumoral, or injected in the vicinity of a tumor.

[0186] In embodiments, the cancer is a blood cancer.

[0187] In some embodiments, the cancer is a solid tumor.

[0188] In various embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.

[0189] Another aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for treating a cancer.

[0190] Yet another aspect of the present disclosure is a method for killing a cancer cell or for inhibiting the proliferation of a cancer cell. The method comprising contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0191] In numerous embodiments, the cancer cell is contacted with the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells simultaneously.

[0192] In embodiments, the cancer cell is contacted with the population of isolated lymphoid lineage cells before being contacted with the population of isolated myeloid lineage cells or the cancer cell is contacted with the population of isolated lymphoid lineage cells after being contacted with the population of isolated myeloid lineage cells.

[0193] In some embodiments, wherein one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR). In some cases, the CAR comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In various cases, the antigen binding region binds to one or more tumor antigens. In these embodiments, the CAR may comprise an antigen binding region that binds to ROR1.

[0194] In various embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine.

[0195] In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, optionally, wherein the cells express a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0196] In embodiments, the isolated lymphoid lineage cells comprise cytotoxic lymphocytes. In some cases, the isolated lymphoid lineage cells comprising cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells. In various cases, the cytotoxic lymphocyte targets and kills cancer cells, e.g., the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. The cytotoxic lymphocyte has reduced cytotoxicity to an NK-resistant cancer cell. In some cases, the cytotoxic lymphocyte is a Natural Killer (NK) cell, e.g., a mature NK cell, or is a cytotoxic T cell. The cytotoxic lymphocyte may be a Natural killer T (NKT) cell. In these embodiments, the NK cell expresses CD16a and / or the NK cell does not express CD3 and / or the NK cell is CD56bright CD16dim / −. In some cases, the NK cell secretes one or more cytokines selected from interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b). In other case, the cytotoxic lymphocyte is a delta-gamma T cell. In these embodiments, the cytotoxic lymphocyte is further engineered to express a chimeric antigen receptor (CAR) and / or is further engineered to express or overexpress a cytokine.

[0197] In some embodiments, the isolated myeloid lineage cells comprise a megakaryocyte, erythrocyte, mast cell, myeloblast, dendritic cell, basophil, neutrophil, eosinophil, monocyte, or macrophage.

[0198] In various embodiments, the isolated myeloid lineage cells express one or more of CD11b, CD13, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., in amounts that are similar to amounts expressed by PBMC-derived cells.

[0199] In numerous embodiments, the isolated myeloid lineage cells have increased expression of CD80 and / or CD206, which is indicative of an activated state.

[0200] In embodiments, the isolated myeloid lineage cell is a macrophage. In some cases, the macrophage expresses one or more of CD11b, CD68, CD80, CD86, CD163, CD206, and SIRPα in amounts that are similar to amounts expressed by PBMC-derived cells and / or secretes one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells. The macrophage may express one or more of CD34, CD44, CD45, CD73, and CD90. In various cases, the macrophages comprise M1 macrophages and / or M2 macrophages. The M1 macrophages and / or M2 macrophages may secrete one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells.

[0201] In some embodiments, the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0202] In various embodiments, the isolated myeloid lineage cell is further engineered to express a chimeric antigen receptor (CAR).

[0203] In numerous embodiments, the isolated myeloid lineage cell is further engineered to express or overexpress a cytokine.

[0204] In embodiments, the isolated lymphoid lineage cells and the isolated myeloid lineage cells are manufactured by a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor. In cases when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated. In these embodiments, the stem cell may be an induced pluripotent stem (iPSC). In some cases, the isolated lymphoid lineage cell and the isolated myeloid lineage cell are derived from the same iPSC. In various cases, the iPSC comprises a genomic modification that expresses a chimeric antigen receptor (CAR) and / or comprises a genomic modification that expresses or over expresses a cytokine. The iPSC may comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, e.g., a biallelic disruption in a B2M gene; in these cases, the iPSC expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0205] In embodiments, the iPSC was reprogrammed from a somatic cell, and the method further comprises contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors. The one or more reprogramming factors may be selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In some cases, the somatic cell is selected from fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0206] In some embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vitro.

[0207] In various embodiments, contacting the cancer cell with a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells occurs in vivo.

[0208] Yet a further aspect of the present disclosure is a plurality of compositions for use in any herein-disclosed method for killing a cancer cell or for inhibiting the proliferation of a cancer cell.

[0209] In an aspect, the present disclosure provides a method for manufacturing a plurality of population of cells comprising a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells for treating a cancer, for killing a cancer cell, and / or for inhibiting the proliferation of a cancer cell. The method a method comprising steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5a) culturing a first subset of the CD34+ cells in a lymphoid progenitor medium and (5b) culturing a second subset of the CD34+ cells in a myeloid progenitor medium; (6a) culturing the cells of step (5a) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes and (6b) culturing the cells of step (5b) in a macrophage cell medium under conditions to obtain a population of cells enriched for macrophages; wherein steps (5a) and (6a) occur in an adherent culturing vessel, and steps (5b) and (6b) occur in a bioreactor.

[0210] In numerous embodiments, when CD34+ cells are selected, the embryoid bodies are first chemically and / or mechanically dissociated.

[0211] In embodiments, the stem cell is an induced pluripotent stem (iPSC). In some cases, the isolated lymphoid lineage cell and the isolated myeloid lineage cell are derived from the same iPSC. The iPSC may comprise a genomic modification that expresses a chimeric antigen receptor (CAR) and / or a genomic modification that expresses or over expresses a cytokine. In various cases, the iPSC comprises a genomic modification which disrupts the beta-2-microglobulin (B2M) gene, e.g., a biallelic disruption in a B2M gene. In these cases, the iPSC expresses a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0212] In some embodiments, the iPSC was reprogrammed from a somatic cell, and the method further comprises contacting the somatic cell with one or more ribonucleic acids (RNAs), wherein each RNA encodes one or more reprogramming factors. The one or more reprogramming factors may be selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In some case, the somatic cell is selected from fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0213] In various embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses a chimeric antigen receptor (CAR), e.g., which comprises an antigen binding region that binds to one or more antigens expressed by a cancer cell. In some case, the antigen binding region binds to one or more tumor antigens. In these embodiments, the CAR may comprise an antigen binding region that binds to ROR1. In numerous embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification that expresses or over expresses a cytokine.

[0214] In embodiments, one or more of the isolated lymphoid lineage cells and / or one or more of the isolated myeloid lineage cells comprise a genomic modification which disrupts the beta-2-microglobulin (B2M) gene. In some cases, the cells express a fusion protein comprising a B2M polypeptide and an HLA polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide).

[0215] In some embodiments, the isolated lymphoid lineage cells comprise cytotoxic lymphocytes. In some cases, the isolated lymphoid lineage cells comprising cytotoxic lymphocytes are enriched for CD56+ cells, for CD16+ cells, NKG2D+ cells, CD226+ Cells, NKp46+ cells, NKp44+ cells, CD244+ cells, and / or CD94+ cells. The cytotoxic lymphocyte targets and kills cancer cells. In various cases, the cytotoxic lymphocyte targets and kills cancer cells without requiring IL-15 and / or without requiring IL-2 activation. The cytotoxic lymphocyte may have reduced cytotoxicity to an NK-resistant cancer cell. The cytotoxic lymphocyte may be a Natural Killer (NK) cell, e.g., a mature NK cell, or is a cytotoxic T cell The cytotoxic lymphocyte may be a Natural killer T (NKT) cell. The the NK cell may express CD16a and / or the NK cell does not express CD3 and / or the NK cell is CD56bright CD16dim / −. In various cases, the NK cell secretes one or more cytokines selected from interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), macrophage inflammatory protein-1a (MIP-1a), and macrophage inflammatory protein-1b (MIP-1b). The cytotoxic lymphocyte may be a delta-gamma T cell. In some cases, the cytotoxic lymphocyte is further engineered to express a chimeric antigen receptor (CAR) and / or is further engineered to express or overexpress a cytokine.

[0216] In various embodiments, the isolated myeloid lineage cells comprise a megakaryocyte, erythrocyte, mast cell, myeloblast, dendritic cell, basophil, neutrophil, eosinophil, monocyte, or macrophage.

[0217] In numerous embodiments, the isolated myeloid lineage cells express one or more of CD11b, CD13, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., in amounts that are similar to amounts expressed by PBMC-derived cells.

[0218] In embodiments, the isolated myeloid lineage cells have increased expression of CD80 and / or CD206, which is indicative of an activated state.

[0219] In some embodiments, the isolated myeloid lineage cell is a macrophage. In some cases, the macrophage expresses one or more of CD11b, CD68, CD80, CD86, CD163, CD206, and SIRPα in amounts that are similar to amounts expressed by PBMC-derived cells and / or secretes one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells. In various cases, the macrophage expresses one or more of CD34, CD44, CD45, CD73, and CD90. In these embodiments, the method may further comprise a step of differentiating the macrophages into M1 and / or M2 macrophages, e.g., by exposure to MCSF. The method may also further comprise a step of polarizing the M1 macrophages with interferon gamma (IFN-γ) and / or lipopolysaccharide (LPS) and / or treating the M2 macrophages with IL-4. In various cases, the macrophages comprise M1 macrophages and / or M2 macrophages; the M1 macrophages and / or M2 macrophages may secrete one or more of TNFα, IL-12p70, and IL-10 in amounts that are similar to amounts expressed by PBMC-derived cells.

[0220] In various embodiments, the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0221] In numerous embodiments, the isolated myeloid lineage cell is further engineered to express a chimeric antigen receptor (CAR).

[0222] In embodiments, the isolated myeloid lineage cell is further engineered to express or overexpress a cytokine.

[0223] In some embodiments, the iPSC was contacted with resveratrol before reprogramming.

[0224] In various embodiments, one or more culturing steps comprise a medium which is serum-free culture medium and / or feeder-free culture medium. In some cases, the serum-free culture medium and / or feeder-free culture medium is an mTeSR™ medium and / or the serum-free culture medium and / or feeder-free culture medium is a StemDiff™ NK medium.

[0225] In numerous embodiments, the adherent culturing vessel is a multi-well plate or a cell culturing flask. In embodiments, the method of manufacturing provides at least 1×106 myeloid lineage cells / ml and at least 3×105 lymphoid lineage cells / ml.

[0226] In some embodiments, the method of manufacturing provides both CD14+ (>95% positive) macrophages and CD56bright / CD16dim NK cells.

[0227] In various embodiments, the method of manufacturing is amenable to scaling to clinically relevant doses.

[0228] In embodiments, the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells act synergistically to kill cancer cells.

[0229] In embodiments, the cancer is a blood cancer.

[0230] In some embodiments, the cancer is a solid tumor.

[0231] In various embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.

[0232] In another aspect, the present disclosure provides a plurality population of cells comprising a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells for treating a cancer, for killing a cancer cell, and / or for inhibiting the proliferation of a cancer cell which were manufactured by any herein-disclosed method.Chimeric Antigen Receptor (CAR)-Bearing Cytotoxic Lymphocytes

[0233] In embodiments, the present cytotoxic lymphocytes are engineered with chimeric antigen receptors (CARs), e.g., the present cytotoxic lymphocytes are CAR-NK cells, CAR-T cells, CAR-myeloid cells, or CAR-mesenchymal stromal / stem cells.

[0234] In embodiments, the cytotoxic lymphocyte, optionally NK cell or T cell, is genetically modified to express a recombinant chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In embodiments, the intracellular signaling domain comprises at least one immune receptor tyrosine-based activation motif (ITAM)-containing domain.

[0235] In embodiments, the intracellular signaling domain is from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0236] In embodiments, the transmembrane domain is from one of CD28 or a CD8.

[0237] In embodiments, the antigen binding region binds one antigen. In embodiments, the binding region binds two antigens.

[0238] In embodiments, the extracellular domain comprising an antigen binding region comprises: (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv). In embodiments, the extracellular domain comprising an antigen binding region comprises two of (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv). In embodiments, the extracellular domain comprising an antigen binding region comprises one of each of: (a) a natural ligand or receptor, or fragment thereof, and (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0239] In embodiments, the antigen binding region binds a tumor antigen.

[0240] In embodiments, the antigen binding region comprises one or more of (i) CD94 / NKG2a, which optionally binds HLA-E on a tumor cell; (ii) CD96, which optionally binds CD155 on a tumor cell; (iii) TIGIT, which optionally binds CD155 or CD112 on a tumor cell; (iv) DNAM-1, which optionally binds CD155 or CD112 on a tumor cell; (v) KIR, which optionally binds HLA class I on a tumor cell; (vi) NKG2D, which optionally binds NKG2D-L on a tumor cell; (vii) CD16 (e.g., CD16a or CD16b), which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; (viii) NKp30, which optionally binds B7-H6 on a tumor cell; (ix) NKp44; and (x) NKp46.

[0241] In embodiments, the antigen binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6, as well as any variant thereof.

[0242] In embodiments, the antigen binding region binds an antigen, e.g., a tumor antigen, selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, as well as any variant thereof. In embodiments, an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, as well as any variant thereof can be used as a single-target CAR, dual-target CAR, mAb, or any combination of any of those.

[0243] In embodiments, the antigen binding region binds two antigen, e.g., two tumor antigens, the antigens being: (a) an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2, as well as any variant thereof and (b) an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2, as well as any variant thereof.

[0244] In embodiments, the antigen binding region binds two antigen, the antigens being: (a) an antigen selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvlll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-1, IGF-I I, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), multiple myeloma or lymphoblastic leukemia antigen, such as one selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5, a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any variant thereof and (b) an antigen selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variantI II (EGFRvl ll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-l, IGF-I I, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), multiple myeloma or lymphoblastic leukemia antigen, such as one selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5, a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any variant thereof.

[0245] In embodiments, the extracellular domain of the recombinant CAR comprises the extracellular domain of an NK cell activating receptor or a scFv.

[0246] In embodiments, the NK cell comprises a gene-edit in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1.

[0247] In embodiments, the gene-edit in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1 is caused by contacting the cell with RNA encoding one or more gene-editing proteins. In embodiments, the gene-edit of causes a reduction or elimination of expression and / or activity of IL-6, NKG2A, NKG2D, KIR, TRAC, PD1, and / or HPK1. In embodiments, the gene-edit causes an increase of expression and / or activity of IL-7, CCL17, CCR4, IL-6R, IL-12, IL-15, and / or TRAIL.

[0248] In embodiments, the cytotoxic lymphocyte, e.g., a T cell, NK cell, further comprises one or more recombinant genes capable of encoding a suicide gene product. In embodiments, the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and an apoptotic signaling protein.

[0249] Any cytotoxic lymphocyte disclosed herein (e.g., manufactured by a method disclosed herein, comprising a gene edit (e.g., in B2M), expressing a high affinity CD16a receptor, and / or expressing a fusion protein comprising B2M polypeptide and an HLA polypeptide) can be further genetically engineered to express a CAR.mRNA Cell Engineering Enables Rapid Prototyping of Macrophage Gene-Editing Strategies for Cancer Immunotherapy Applications

[0250] Macrophages' ability to infiltrate solid tumors and engage in both direct killing of cancer cells and recruitment of other immune cells has made them a promising target for development of next-generation cancer immunotherapies. The innate ability of macrophages to ingest foreign genetic material also facilitates their engineering with formulated nucleic acids, including mRNA. The oncoantigen tyrosine-protein kinase transmembrane receptor ROR1 has garnered interest for its minimal expression in healthy adult cells and overexpression in many malignancies, including solid tumors associated with ovarian, lung, and triple-negative breast cancer.

[0251] In this disclosure, e.g., in Example 10, an mRNA-based platform for rapid prototyping of macrophage engineering approaches is described. Here is shown mRNA delivery to peripheral blood mononuclear cell (PBMC) and iPS cell-derived macrophages for gene editing prototyping and functional assessment of encoded proteins. To develop this platform, macrophages were transfected with unmodified or 5′-methoxyuridine (5-moU)-containing mRNA encoding green fluorescent protein (GFP). Both mRNAs resulted in more than 95% of cells displaying GFP within 4 hours. ROR1-targeting CAR with a CD3 zeta activation domain and 4-1BB costimulatory domain was designed.

[0252] Transfecting mRNA encoding the ROR1-CAR yielded 70% CAR-expressing cells, as measured using PE-labelled ROR1. The ROR1 affinity of rabbit and humanized binding domains was analyzed and the humanized binding domain displayed a 2.5-fold increase in affinity as measured by flow cytometry using PE-labelled ROR1. The human receptor domain, but not the rabbit domain, demonstrated activation when bound to ROR1 as assessed by immunofluorescence of CD3 zeta phosphorylation. The mRNA-encoded ROR1-CAR's functionality was assayed by measuring killing of ROR1-expressing SKOV-3 ovarian cancer cells. Both the rabbit and humanized ROR1 domains of the CAR displayed significantly increased cytotoxicity towards SKOV-3 cells when compared with un-transfected macrophages after a 24-hour co-culture at a 5:1 effector-to-target ratio (p<0.01). The ROR1-CAR sequence was inserted into the AAVS1 safe harbor locus of iPSCs under the control of an SFC promotor, isolated5 biallelic-inserted lines, and the resulting cells were differentiated into macrophages. These results demonstrated an mRNA-based platform for rapid prototyping of macrophage engineering approaches. Transfection of macrophages with mRNA encoding a chimeric antigen receptor (CAR) targeting ROR1 resulted in functional expression in vitro, facilitating optimization of the antibody and co-stimulatory domains to improve protein binding affinity and immune activation. This platform thus enables the assessment and validation of novel macrophage gene editing strategies and is being explored for the development of macrophage-engineering therapies for solid tumor applications.

[0253] Indeed, this method permits screening of a library of CAR constructs in vitro to determine which constructs are readily expressed and which are most functional, e.g., in targeting and / or killing cancer cells. Once a CAR construct has been identified as superior, cells ex vivo may be gene-edited to express the construct and the edited cells may be administered into a subject in need and / or the cells of the subject in need may be gene-edited in vivo such that the construct genetically modify cells within the subject.

[0254] An aspect of the present disclosure is a method for screening constructs capable of being expressed in an in vivo cell and for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; and (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell. Another aspect of the present disclosure is method for screening constructs capable of being expressed in an ex vivo cell and for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) culturing the cell capable of expressing the fusion protein which recognizes and / or binds to a cancer cell until a therapeutic amount of the cell is manufactured.

[0255] A further aspect of the present disclosure is a method for screening constructs capable of being expressed in an ex vivo cell and for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) contacting an ex vivo cell with the synthetic mRNA encoding the gene-editing protein and the repair template encoding the fusion protein which was identified in step (3) as having the ability recognize and / or bind to a cancer cell; and (5) culturing the cell of step (4) until a therapeutic amount of the cell is manufactured.

[0256] An additional aspect of the present disclosure is a method for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; and (4) administering to a subject in need the synthetic mRNA encoding the gene-editing protein and the repair template encoding the fusion protein which has the ability recognize and / or bind to a cancer cell.

[0257] In an aspect, the present disclosure provides a method for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) culturing the cell capable of expressing the fusion protein which recognizes and / or binds to a cancer cell until a therapeutic amount of the cell is manufactured; and (5) administering a therapeutically-effective amount of the cells of step (4) to a subject in need. In another aspect, the present disclosure provides a method for treating a cancer. The method comprising: (1) obtaining a cultured cell which corresponds to cell type present in a subject; (2) transfecting the cultured cell with a synthetic mRNA encoding a gene-editing protein and a repair template encoding a fusion protein that recognizes and / or binds to a cancer cell; (3) identifying cells that have been transformed and capable of expressing the fusion protein for the ability of its fusion protein to recognize and / or bind to a cancer cell; (4) contacting an ex vivo cell with the synthetic mRNA encoding the gene-editing protein and the repair template encoding the fusion protein which was identified in step (3) as having the ability recognize and / or bind to a cancer cell; (5) culturing the cell of step (4) until a therapeutic amount of the cell is manufactured; and (6) administering a therapeutically-effective amount of the cells of step (4) to a subject in need.

[0258] In embodiments, the fusion protein that recognizes and / or binds to a cancer cell is a chimeric antigen receptor (CAR).

[0259] In embodiments, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region.

[0260] In embodiments, the intracellular signaling domain comprises at least one immunoreceptor tyrosine-based activation motif (ITAM)-containing domain.

[0261] In embodiments, the intracellular signaling domain is from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0262] In embodiments, the transmembrane domain is from one of CD28 or a CD8.

[0263] In embodiments, the antigen binding region binds one antigen.

[0264] In embodiments, the antigen binding region binds two antigens.

[0265] In embodiments, the extracellular domain comprising an antigen binding region comprises: (a) C natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0266] In embodiments, the extracellular domain comprising an antigen binding region comprises two of (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0267] In embodiments, the extracellular domain comprising an antigen binding region comprises one of each of: (a) a natural ligand or receptor, or fragment thereof, and (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0268] In embodiments, the antigen binding region binds a tumor antigen.

[0269] In embodiments, the antigen binding region comprises one or more of: a. CD94 / NKG2a, which optionally binds HLA-E on a tumor cell; b. CD96, which optionally binds CD155 on a tumor cell; c. TIGIT, which optionally binds CD155 or CD112 on a tumor cell; d. DNAM-1, which optionally binds CD155 or CD112 on a tumor cell; e. KIR, which optionally binds HLA class I on a tumor cell; f. NKG2D, which optionally binds NKG2D-L on a tumor cell; g. CD16a, which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; h. NKp30, which optionally binds B7-H6 on a tumor cell; i. NKp44; and j. NKp46.

[0270] In embodiments, the antigen binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6.

[0271] In embodiments, the antigen binding region binds an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2.

[0272] In embodiments, the antigen binding region binds two antigens, the antigens being: a. an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2 and b. an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2.

[0273] In embodiments, the extracellular domain of the recombinant CAR comprises the extracellular domain of an NK cell activating receptor or a scFv.

[0274] In embodiments, the cell type is of the lymphoid cell lineage or the myeloid cell lineage. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell, or an NK cell, e.g., an NK-T cell. In various cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0275] In embodiments, after gene editing, the cell is a CAR-T cell, CAR-NK cell, a CAR-myeloid cell, or a CAR-mesenchymal stromal / stem cell.

[0276] When gene-editing is performed in vivo, transfection may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System, e.g., which relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.FIG. 30 is a cartoon showing methods for rapid prototyping of macrophage gene-editing strategies for cancer immunotherapies.Immune Silenced, Yet Self-Activating, Proliferative, and Anti-Tumoral Cells

[0278] Autologous engineered cell therapies such as autologous chimeric antigen receptor T-cell (CAR-T) therapies have revolutionized the treatment of hematologic cancers, however they are limited by manufacturing time and variability, the requirement for lymphodepletion, and side effects related to cytokine release. Allogeneic cell therapies derived from gene-edited induced pluripotent stem cells (iPSCs) are being developed to address the challenges associated with autologous engineered cell therapies. These “off-the-shelf” cell therapies contain specific edits designed to reduce immune rejection and to confer enhanced therapeutic properties and greater safety. However, efficient, footprint-free, biallelic targeting of defined loci in iPSCs remains technically challenging with current gene-editing approaches.

[0279] Further, while induced pluripotent stem cells (iPSCs) readily differentiate into a wide variety of cell types both in vitro and in vivo, the development of directed differentiation protocols that reliably yield pure populations of functional cells has proved challenging, in particular when differentiating into cell of the lymphoid or myeloid lineage. Generating functional cytotoxic lymphocytes from iPSCs is of particular interest to support the development of off-the-shelf engineered cell therapies for immune-oncology applications.

[0280] What is needed is improved compositions and methods for generating cellular therapies that can be engineered and produced in a practical manner.

[0281] The present disclosure is based, in part, on the discovery that cytotoxic lymphocytes, of the lymphoid cell lineage, e.g., T cells, NK cells, or cells of the myeloid lineage, e.g., macrophages, or mesenchymal stromal / stem cells, or hematopoietic stem cells can be gene-edited and differentiated, using mRNA- and iPS-based methods, to yield therapeutic cells that are immune silenced, yet self-activating, proliferative, and anti-tumoral.

[0282] Cytotoxic lymphocytes, including T cells and NK cells, are being developed as allogeneic, “off-the-shelf”, cell therapies for the treatment of hematological and solid tumors. Allogenic lymphocyte therapies face challenges, however, including limited expansion potential and limited in vivo persistence due to host immune rejection.

[0283] The resulting lymphocytes were characterized for surface markers via flow cytometry and incubated with cancer cells to assess tumor cell engagement and cytotoxicity. Notably, consistently higher yields of lymphocytes were obtained from the B2M-knockout iPSC line relative to a parental, wild-type iPSC line. Both wild-type and B2M-knockout lymphocytes cells killed 75-90% of K562 cells after 24 hours (effector to target (E:T) ratio of 5:1). Interestingly, cytotoxic lymphocytes derived from B2M-knockout iPSCs exhibited greater K562 cell killing with the addition of IL15 and IL2, while killing by wild-type cells was not controlled by these activating cytokines. Cancer cell killing activity was maintained through cryopreservation, albeit at a reduced level (15-40% reduction in activity). Accordingly, B2M-knockout iPSCs of the present disclosure are an ideal source of cytotoxic lymphocytes for the development of “off-the-shelf” allogeneic cell therapies for the treatment of cancer and without substantial host immune rejection.

[0284] In one aspect, there is provided cell comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a loss of function, optionally in both alleles, of the B2M gene, wherein the cell is a cytotoxic lymphocyte from a lymphoid lineage cell or the cell is a myeloid lineage cell. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage. In embodiments the cytotoxic lymphocyte is a NK cell.

[0285] The present cytotoxic lymphocyte is sometimes referred to herein as an “engineered cytotoxic lymphocyte”.

[0286] An aspect of the present disclosure is a method for manufacturing a population of cells that is enriched for cytotoxic lymphocytes. The method comprises steps of: (1) obtaining a stem cell; (2) culturing the stem cell in a bioreactor comprising a media that promotes formation of spheroids; (3) culturing the spheroids in a bioreactor in a media that promotes formation of embryoid bodies; (4) optionally, selecting CD34+ cells from the embryoid bodies; (5) culturing the CD34+ cells in a lymphoid progenitor medium; and (6) culturing the cells of step (5) in an NK cell medium under conditions to obtain a population of cells enriched for cytotoxic lymphocytes. In this aspect, steps (5) and (6) occur in an adherent culturing vessel. When CD34+ cells are selected, the embryoid bodies may be first chemically and / or mechanically dissociated. In embodiments, the stem cell is an induced pluripotent stem (iPSC). In this aspect, the stem cell has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene. In some cases, the stem cell has a biallelic disruption in a B2M gene.

[0287] In another aspect, there is provided a method of making an engineered cell comprising a disrupted B2M gene, the method, comprising (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins; and (c) differentiating the iPS cell into a cytotoxic lymphocyte, e.g., a cell of the lymphoid cell lineage or into a cell of the myeloid cell lineage. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0288] An aspect of the present disclosure is a method for killing a cancer cell. The method comprising steps of: (1) obtaining a herein-disclosed cytotoxic lymphocyte which was derived from a stem cell has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a biallelic B2M knockout, and (2) contacting cytotoxic lymphocyte with the cancer. In some cases, the cancer cell is in vivo.

[0289] Yet another aspect of the present disclosure is a method for treating a cancer patient in need thereof. The method comprising a step of administering to the cancer patient a therapeutically-effective amounts of a herein-disclosed cytotoxic lymphocyte which was derived from a stem cell has a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a biallelic B2M knockout.

[0290] In some cases, mRNA-reprogrammed iPSC lines with a biallelic knockouts of the beta-2 microglobulin (B2M) gene, a key component of MHC class I molecules, are obtained using an mRNA-encoded chromatin context-sensitive gene-editing endonuclease. The B2M-knockout iPSCs may be differentiated using a novel, fully suspension process that replaces specialized micropatterned culture vessels with a spheroid culture step. Additional details regarding B2M knockout iPSCs useful in the present disclosure are described in PCT / US2022 / 019020, the contents of which are incorporated herein by reference in its entirety.

[0291] In another aspect, there is provided a method of treating cancer, comprising (a) obtaining an isolated cytotoxic lymphocyte comprising a genetically engineered disruption in a B2M gene; and (b) administering the isolated cytotoxic lymphocyte to a subject in need thereof, wherein the cytotoxic lymphocyte is selected from a lymphoid cell or a myeloid cell. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.Immune Silencing

[0292] In embodiments, the present cytotoxic lymphocyte is engineered to evade recognition and / or clearance by a host immune system. In embodiments, the present cytotoxic lymphocyte is a stealth cytotoxic lymphocyte. In embodiments, the present cytotoxic lymphocyte is not substantially recognized by an immune system upon administration to a subject.

[0293] In embodiments, the present cytotoxic lymphocyte has reduced or eliminated susceptibility to cell killing by T cells as compared to a cytotoxic lymphocyte which does not comprise a genetically engineered disruption in the B2M gene. In embodiments, the present cytotoxic lymphocyte has reduced or eliminated susceptibility to cell killing by other cytotoxic lymphocytes as compared to another cytotoxic lymphocyte which comprises a genetically engineered disruption in the B2M gene. In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of B2M is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of B2M is reduced or inhibited.

[0294] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of MHC class I is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of MHC class I is reduced or inhibited.

[0295] In embodiments, the B2M gene is a human B2M gene (e.g., NCBI Reference Sequence: NG_012920). The sequence of the B2M gene of various embodiments is provided in the EXAMPLES section herein. B2M, is the light chain of MHC class I molecules, and as such an integral part of the major histocompatibility complex. In humans, B2M is encoded by the b2m gene which is located on chromosome 15. The human protein is composed of 119 amino acids and has a molecular weight of 11.8 kilodaltons (e.g., UniProtKB—P61769). The amino acid sequence of human beta-2-microglobulin (B2M) is:(SEQ ID NO: 13)MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.

[0296] In embodiments, the present cytotoxic lymphocyte has genetically engineered disruptions of all substantially all copies of the B2M gene. In embodiments, the present cytotoxic lymphocyte has a loss of function of the B2M gene. In embodiments, the present cytotoxic lymphocyte has a loss of function of both alleles of the B2M gene.

[0297] In embodiments, the genetically engineered disruption of the B2M gene is in exon 3 of human B2M. In embodiments, the genetically engineered disruption of the B2M gene is a deletion. In embodiments, the deletion is about 10 to about 20 nucleotides. In embodiments, the deletion is near nucleotides 500 to 550 of the human B2M gene. In embodiments, the deletion is of the sequence TTGACTTACTGAAG (SEQ ID NO: 14), or a functional equivalent thereof.

[0298] In embodiments, the present cytotoxic lymphocyte has downregulated MHC class I expression and / or activity.

[0299] In embodiments, the genetically engineered disruption of B2M comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins.

[0300] In embodiments, the present cytotoxic lymphocyte is engineered to be further immune silenced, e.g., in addition to B2M (MHC Class I) disruption. In embodiments, the present cytotoxic lymphocyte is engineered to be disrupted at the human MHC II transactivator (CIITA) gene (NCBI Reference Sequence: NG_009628.1).

[0301] In embodiments, the present cytotoxic lymphocyte has downregulated MHC class II expression and / or activity.

[0302] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of CIITA is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of CIITA is reduced or inhibited.

[0303] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of MHC class II is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of MHC class II is reduced or inhibited.

[0304] In embodiments, the genetically engineered disruption of CIITA comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins.

[0305] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of B2M and CIITA is reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of B2M and CIITA is reduced or inhibited.

[0306] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of MHC class I and MHC class II are reduced or inhibited. In embodiments, the present cytotoxic lymphocyte is characterized in that the function of MHC class I and MHC class II are reduced or inhibited.

[0307] In embodiments, the genetically engineered disruption of B2M and CIITA comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins. In embodiments, the present cytotoxic lymphocyte comprises a genetically engineered alteration in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0308] In embodiments, the cytotoxic lymphocyte expresses a fusion protein comprising a B2M polypeptide and a HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G polypeptide. The fusion protein may be expressed by insertion of a repair template into a single or double strand break of the B2M gene; in some cases, the repair template comprises the coding sequence for B2M and the HLA gene. Notably, the fusion protein replaces endogenous B2M and HLA pairs expressed by a cytotoxic lymphocyte, thereby reducing the likelihood that the cytotoxic lymphocyte will be reduced or eliminated by a host cytotoxic lymphocyte.

[0309] In embodiments, the present cytotoxic lymphocyte does not comprise a genetically engineered alteration in one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0310] In embodiments, the genetically engineered alteration is a genetically engineered reduction or elimination in expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0311] In embodiments, the genetically engineered alteration is a genetically engineered increase in expression and / or activity of one or more genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0312] In embodiments, the genetically engineered disruption of B2M is combined with a genetically engineered expression of a fusion between B2M or a fragment thereof and one or more genes and / or fragments thereof selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.

[0313] In embodiments, the B2M or fragment thereof and one or more genes and / or fragments thereof are separated by a linker region. In embodiments the linker is (G4S)3.

[0314] In embodiments, the genetically engineered alteration is a genetically engineered increase in expression and / or activity of one or more genes selected from IL-2, IL-15, IL-21. In embodiments, the IL-15 contains the N72D mutation. In embodiments, the IL-15 is fused to the cytokine binding domain of IL-15Rα.

[0315] In embodiments, the present cytotoxic lymphocyte is characterized in that the expression of negative regulators of IL-15 signaling are reduced or inhibited. In embodiments, the negative regulator of IL-15 signaling is the CISH protein. In embodiments, the reduction or inhibition of negative regulators of IL-15 signaling is achieved by genetically engineered disruption of the CISH gene. The Cytokine-inducible SH2-containing protein (CISH) gene is found at gene ID: NG_023194.1.

[0316] In embodiments, the genetically engineered disruption of CISH comprises a gene-edit and the gene-edit is caused by contacting the cell with RNA encoding one or more gene-editing proteins.Methods of Making B2M Knockout Cells

[0317] In aspects, the present disclosure provides a method of making an engineered cell comprising a disrupted B2M gene, the method comprising: (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a beta-2-microglobulin (B2M) gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins; and (c) differentiating the iPS cell into a cytotoxic lymphocyte, e.g., a cell of the lymphoid cell lineage, or into a cell of the myeloid cell lineage, or a mesenchymal stem / stromal cell, or a hematopoietic stem cell. In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0318] In embodiments, the method further comprises disrupting a CIITA gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene-editing proteins.

[0319] In embodiments, the cytotoxic lymphocyte is an NK cell.

[0320] In embodiments, the somatic cell is a fibroblast or keratinocyte.

[0321] In embodiments, the method provides an increased proliferation rate of iPS cells as compared to the rate of iPS cells without a disruption of the B2M gene.

[0322] In embodiments, the method provides an increased proliferation rate of differentiating cells along a lymphoid lineage cell as compared to the rate of iPS cells without a disruption of the B2M gene.

[0323] In embodiments, the method provides an increased expansion of differentiating cells along a lymphoid lineage cell as compared to the rate of iPS cells without a disruption of the B2M gene.

[0324] In embodiments, the differentiating comprises embryoid body-based hematopoietic commitment. In embodiments, the differentiating comprises enrichment of CD34+ cells. In embodiments, the differentiating comprises differentiating into CD5+ / CD7+ common lymphoid progenitors.

[0325] In embodiments, the method yields CD56dim CD16+ NK cells.

[0326] In embodiments, the RNA is associated with one or more lipid selected from and / or Formulae I-XVI.Differentiation of iPSCs into Monocytes and Macrophages

[0327] In one aspect, the present disclosure relates to induced pluripotent stem cell (iPSC)-derived monocytes that can be differentiated into functional M1 and M2 macrophages with enhanced cytokine secretion and tumor cell-killing activity.

[0328] Although cancer immunotherapy has advanced rapidly over the past two decades, with several autologous chimeric antigen receptor (CAR)-T cell therapies approved for the treatment of hematologic cancers, CAR-T cells have shown limited activity against solid tumors, in part due to the immunosuppressive nature of the tumor microenvironment preventing CAR-T cell infiltration. This has led to investigation of other immune cells as alternatives to T-cell-based therapies, including monocytes and monocyte-derived macrophages, which exhibit innate tumor-infiltration properties. Disclosed herein is a new and useful process for differentiating pluripotent stem cells along a myeloid lineage, and generated populations of cells with characteristics of monocytes and M1 and M2 macrophages, including cytokine secretion and tumor cell-killing activity. mRNA-reprogrammed human induced pluripotent stem cells (iPSCs) were differentiated into monocytes using a 28-day monolayer protocol. Beginning on day 14, cells were harvested every 3-4 days. CD14+ isolation yielded >95% CD14+ cells with an average yield of 4.1×104 cells per cm2 per harvest. iPSC-derived monocytes were compared to peripheral blood mononuclear cell (PBMC)-derived monocytes for expression of key hematopoietic and myeloid-lineage markers CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα. iPSC-derived monocytes showed similar expression of CD11b, CD14, CD33, CD45, and CD163 compared to PBMC-derived monocytes, and increased expression of markers indicative of an activated state: CD80 and CD206. Compared to PBMC-derived monocytes, iPSC-derived monocytes showed both higher viability in culture and superior recovery from cryopreservation. iPSC-derived monocytes were further differentiated into macrophages by exposure to MCSF for 3-4 days, and were assessed for their ability to polarize, secrete pro- and anti-inflammatory cytokines, and for cytotoxic activity when co-cultured with cancer cells. M1 macrophages were polarized with interferon gamma (IFN-γ, 50 ng / mL) and lipopolysaccharide (LPS, 10 ng / mL) for 48 hours, while M2 macrophages were treated with IL-4 (10 ng / mL) for 48 hours. iPSC-derived monocytes differentiated into macrophages with >90% efficacy, as assessed by cell adherence, morphology, and surface marker expression (CD14, CD45, CD163). M1 and M2 polarized iPSC-derived macrophages secreted similar levels of TNFα, IL-12p70, and IL-10 compared to PBMC-derived macrophages. iPSC-derived macrophages killed 45% of U2OS cancer cells in vitro after 24 hours at an E:T ratio of 5:1. Disclosed herein is a process for differentiating mRNA-reprogrammed iPSCs into cytotoxic macrophages. The mRNA reprogramming and differentiation processes are virus-free and DNA-free, avoiding any potential risk of vector integration. This disclosure provides proof of concept that mRNA-reprogrammed iPSCs represent a viable source of macrophages for the development of therapies to treat various indications, including solid tumors. The process is illustrated in FIG. 42.

[0329] Monocytes are a type of leukocyte, or white blood cell. They are the largest type of leukocyte and can differentiate into macrophages and conventional dendritic cells. As a part of the vertebrate innate immune system monocytes also influence adaptive immune responses and exert tissue repair functions. There are at least three subclasses of monocytes in human blood based on their phenotypic receptors: The classical monocyte is characterized by high level expression of the CD14 cell surface receptor (CD14++ CD16− monocyte), the non-classical monocyte shows low level expression of CD14 and additional co-expression of the CD16 receptor (CD14+CD16++ monocyte), and the intermediate monocyte expresses high levels of CD14 and low levels of CD16 (CD14++CD16+ monocytes).

[0330] Monocytes are mechanically active cells and migrate from blood to an inflammatory site to perform their functions. In general, monocytes and their macrophage and dendritic cell progeny serve three main functions in the immune system: these are phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of microbes and particles followed by digestion and destruction of this material. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter.

[0331] Macrophages engulf and digest pathogens, such as cancer cells, microbes, cellular debris, and foreign substances, which do not have proteins that are specific to healthy body cells on their surface, via phagocytosis.

[0332] Macrophage that encourages inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages. M1 macrophages are classically activated, typically by IFN-γ or lipopolysaccharide (LPS), and produce proinflammatory cytokines, phagocytize microbes, and initiate an immune response. M1 macrophages produce nitric oxide (NO) or reactive oxygen intermediates (ROI) to protect against bacteria and viruses. M2 macrophages are alternatively activated by exposure to certain cytokines such as IL-4, IL-10, or IL-13. M2 macrophages will produce either polyamines to induce proliferation or proline to induce collagen production. These macrophages are associated with wound healing and tissue repair. iPSCs obtained by a herein-disclosed method may be differentiated into a monocyte which can be further differentiated into a macrophage, e.g., an M1 or M2 macrophage.

[0333] The general steps for this process comprise (1) iPSC to monocyte differentiation, (2) CD14+ magnetic bead positive selection, and (3) monocyte to macrophage differentiation. These general steps are, respectively, shown in FIG. 43A, FIG. 43B, and FIG. 43C.

[0334] As shown in FIG. 43A, embryonic stem cells (ES cells) may be used as starting material for the process rather than iPSCs.

[0335] Beginning on day 14, monocyte cells can be harvested and continue to be harvested every 3-4 days. Additional details regarding differentiation of iPSCs to monocytes is found at the World Wide Web (www) stemcell.com / stemdiff-monocyte-kit.html, the contents of which are incorporated herein by reference in its entirety.

[0336] As shown in FIG. 43B, CD14+ cells, which comprise monocytes and macrophages, are separated using suitable reagents.

[0337] Finally, as shown in FIG. 43C, monocytes are cultured under conditions such that they differentiate into macrophages, e.g., by activation with macrophage colony-stimulating factor (M-CSF). Additional details regarding differentiation of monocytes to macrophage is found at the World Wide Web (www) stemcell.com / immunocult-sf-macrophage-medium.html, the contents of which are incorporated herein by reference in its entirety.

[0338] M1 macrophages can be then polarized into M1 macrophages with interferon gamma (IFN-γ, 50 ng / mL) and lipopolysaccharide (LPS, 10 ng / mL) for 48 hours, whereas macrophages are treated with IL-4 (10 ng / mL) for 48 hours to generate M2 macrophages.

[0339] Mature differentiated cells can be reprogrammed and dedifferentiated into embryonic-like cells, with embryonic stem cell-like properties. Fibroblast cells can be reversed into pluripotency via, for example, retroviral transduction of certain transcription factors or transfection of synthetic nucleic acids encoding transcription factors, resulting in iPSCs. In some embodiments, iPSCs are generated from various tissues, including fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells. In some embodiments, iPSCs are generated via transfection of synthetic nucleic acids encoding the transcription factors Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein and biologically active fragments, analogues, variants and family-members thereof. In some embodiments, iPSCs are generated via transfection of synthetic nucleic acids encoding miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0340] The generation of iPSCs depends on the transduction of specific transcription factors into the somatic cell genome via vectors for its reprogramming.

[0341] In various cases, transfection of a cell with synthetic nucleic acids for reprogramming may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In some embodiments, following iPSC generation, cells are assessed via pluripotency assays, including morphological and histological analysis, and certain gene expression profiles, proving their ability to differentiate into tissues derived from the three germ layers and teratoma formation. In some embodiments, teratoma assays involve injection of iPSCs into immunocompromised experimental animals and subsequent formed tissue analysis to assess teratoma formation.

[0343] In embodiments, the iPSC is derived from a human. In embodiments, the iPSC is derived from a subject who is not intended to receive the therapy. In embodiments, the iPSC is allogeneic to a patient intended to receive the therapy. In embodiments, the iPSC is from a master cell bank.

[0344] Stem cells have the ability to self-renew and differentiate into multiple cell types and so have applications in regenerative medicine.

[0345] In embodiments, the iPSCs, the monocytes, and / or the macrophage is further gene-edited, as disclosed herein. In some embodiments, the iPSC was gene-edited contemporaneously with being reprogrammed, e.g., from a fibroblast. In various embodiments, the iPSC was gene-edited before being reprogrammed, e.g., from a fibroblast. In other embodiments, the iPSC was gene-edited after being reprogrammed, e.g., from a fibroblast.

[0346] In some aspects of the present disclosure, a macrophage that is administered to a patient, e.g., for killing cancer cell, was ultimately derived from the patient. That is to say, a keratinocyte (as an example) is obtained from the patient and this keratinocyte is reprogrammed into an IPSC which is differentiated into a monocyte and further differentiated into an M1 or M2 macrophage. By starting with the patient's own cells, the macrophage administered back to the patient could be considered to be autologous.

[0347] The use of autologous macrophages in therapeutic applications is safe because the cells will not elicit an immune response. However, it may be difficult to obtain a large amount of bone marrow or adipose tissue from the subject. Autologous macrophages may also have reduced therapeutic efficacy resulting in poor clinical outcomes. Additionally, if macrophages are needed urgently, there may not be time to extract and expand autologous macrophages from a subject.

[0348] Thus, the use of allogeneic macrophages is an attractive alternative because donors can be prescreened for having cells with a high therapeutic potential. Allogeneic macrophages can be prepared on a clinical scale, assayed for therapeutic potential after production and stored in usable clinical doses that can be used readily for urgent therapeutic applications. In some embodiments, the present macrophages are allogeneic.

[0349] Thus, obtaining macrophages by reprogramming iPSCs, which may be obtained by reprogramming human somatic cells, is an attractive alternative. In some embodiments, iPSCs are obtained monocytes are generated via cell reprogramming with non-immunogenic messenger RNA (mRNA) encoding one or more reprogramming factors in a defined, animal-component-free process. The monocytes are further differentiated into macrophages. See, e.g., FIG. 42.

[0350] In some embodiments, monocytes and / or macrophages are checked for safety using one or more of bacterial and fungal tests, Mycoplasma test, adventitious viral agent test, and tumorigenicity assay (karyotype analysis, teratoma formation assay, soft agar assay, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR)).

[0351] In embodiments, the monocyte or macrophage has been altered to reduce expression of MHC molecules. In embodiments, the alteration is enabled by gene editing. In embodiments, the MHC molecules are MHC class I molecules. In embodiments, the expression of MHC class I molecules is reduced by gene editing the B2M gene. In some cases, the gene editing occurs in an iPSC cell that is a progenitor of the monocyte or macrophage.The Immunosuppressive TTAGGG Motif Improves Homology-Directed Insertion of DNA Sequences in Human Primary and Induced Pluripotent Stem (iPS) Cells

[0352] In another aspect, the present disclosure relates to use of TTAGGG motif for decreasing synthetic oligodeoxynucleotides (ODNs)-related activation of pro-inflammatory responses; with decreasing the pro-inflammatory responses leading to higher transgene insertion efficiency.

[0353] Double stranded synthetic oligodeoxynucleotides (dsODNs) have been used as repair templates in gene-editing applications to insert transgenic sequences into defined genomic loci, albeit with low efficiency. Cells engineered in this way are of interest for many therapeutic applications, including allogeneic NK and T cells engineered to express stealthing proteins, cytokines, and chimeric antigen receptors (CARs) for the treatment of a variety of cancers. To increase the efficiency of integration, gene-editing proteins can be co-expressed to create a double-strand break at the target locus. However, recognition of dsODNs by pattern recognition receptors activates signaling cascades resulting in the production of cytokines, including type I interferons such as IFIT1-3 and IFN-β. This immune response can lead to cell cycle arrest, differentiation, and apoptosis and may contribute to low insertion efficiency observed in primary and iPS cells. It has been shown in human immune cells that co-delivery of a short ODN comprising the immunosuppressive motif, TTAGGG, which is found in mammalian telomeric DNA, inhibits the activation of the damage-associate molecular pattern (DAMP) pathway in response to cytosolic DNA. This ODN competitively binds to inflammasomes and reduces the secretion of proinflammatory cytokines. The present disclosure relates to dsODNs comprising the TTAGGG motif for decreasing dsODN-related activation of a pro-inflammatory response in human cells, with decreasing the pro-inflammatory responses leading to higher transgene insertion efficiency. As disclosed herein, the TTAGGG motif is incorporated either at the 5′ end of dsODNs or delivered separately on a short single-stranded ODN (A151). Human primary fibroblasts, iMSCs and iPSCs were electroporated with a dsODN encoding a GFP reporter and containing an SfoI restriction site. Upregulation of pro-inflammatory markers including IFIT1-3, was measured by RT-PCR. A 29-fold higher expression of IFIT1 and IFIT3 was observed in cells electroporated with dsODNs than in untreated controls. Interestingly, including TTAGGG motifs at the 5′-ends of the dsODNs limited the upregulation of IFIT1 and IFIT3 to 10- and 15-fold, respectively, while co-delivery of the TTAGGG motif prevented their upregulation altogether. A gene-editing endonuclease targeting the AAVS1 safe-harbor locus on chromosome 19 was then used to investigate the impact of the TTAGGG motif on the insertion of transgenes at this site. The TTAGGG motif (whether incorporated in the dsODN or co-transfected in the form of the A151 ODN) resulted in approximately 50% higher viability and approximately 50% more GFP-positive cells than when the motif was not present. This disclosure provides proof of concept that the herein-disclosed immunosuppressive sequences increases ODN insertion efficiency and improves cell viability and is therefore a powerful tool for therapeutic knock-in applications, including the generation of knock-in iPS cell lines.

[0354] The process of including a TTAGGG motif in an dsODN for reducing an immune response is illustrated in FIG. 54. As shown in FIG. 54, the TTAGGG motif, found in telomeric DNA, is incorporated onto the 5′ end of a double stranded repair template in primary, iMS and iPS cells. The motif is recognized by, and competitively binds to Pattern Recognition receptors in the cytoplasm of the cells to lessen the immune response mounted against the double stranded repair templates.

[0355] The TTAGGG motif may be provided at the 5′ end of a dsODN that serves as a repair template. The TTAGGG motif may be provided at the 3′ end of a dsODN that serves as a repair template. The TTAGGG motif may be provided at both the 3′ end and the 5′ end of a dsODN that serves as a repair template. The dsODN that serves as a repair template may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the TTAGGG motif.

[0356] As disclosed-herein, the TTAGGG motif may be provided to a cell separately from the dsODN on a short single-stranded synthetic oligodeoxynucleotides (e.g., A151). The A151 ssODN comprise four repeats of the TTAGGG motif and the sequence of TTAGGGTTAGGGTTAGGGTTAGGG (SEQ ID NO: 15). In other cases, a single-stranded synthetic oligodeoxynucleotide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the TTAGGG motif. In some case, the TTAGGG motif may be provided to a cell separately from the dsODN on a short double-stranded synthetic oligodeoxynucleotides. In embodiments, the double-stranded synthetic oligodeoxynucleotide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the TTAGGG motif.

[0357] In some embodiments, the cells that are transfected with a TTAGG-containing ODN (either as a repair template or as separate ODN) are skin cells, pluripotent stem cells, embryonic stem cells, iPSCs, MSCs (including iMSCs), mesenchymal stromal / stem cells, hematopoietic cells, hematopoietic stem cells, lymphocytes, β-cells, T-cells (including CAR-T), NK cell (including CAR-NK), monocytes, macrophages (including CAR-myeloid cells and CAR-mesenchymal stromal / stem cells), retinal pigmented epithelial cells, hematopoietic cells, a hematopoietic stem cells, myeloid cells, tumor-infiltrating lymphocytes, marrow-infiltrating lymphocytes, a peripheral blood lymphocytes, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells.

[0358] In embodiments, gene-editing a cell comprises contacting the cell with synthetic nucleic acid encoding one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyz (SEQ ID NO: 16), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.

[0359] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17) or LTPvQVVAIAwxyzGTHG (SEQ ID NO: 18) and is from 36 to 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20) and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In some embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C.

[0360] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 22) and is from 36 to 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), α is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, α is selected from GHGG (SEQ ID NO: 31), HGSG (SEQ ID NO: 32), HGGG (SEQ ID NO: 33), GGHD (SEQ ID NO: 34), GAHD (SEQ ID NO: 35), AHDG (SEQ ID NO: 36), PHDG (SEQ ID NO: 37), GPHD (SEQ ID NO: 38), GHGP (SEQ ID NO: 39), PHGG (SEQ ID NO: 40), PHGP (SEQ ID NO: 41), AHGA (SEQ ID NO: 42), LHGA (SEQ ID NO: 43), VHGA (SEQ ID NO: 44), IVHG (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), RHGD (SEQ ID NO: 47), RDHG (SEQ ID NO: 48), RHGE (SEQ ID NO: 49), HRGE (SEQ ID NO: 50), RHGD (SEQ ID NO: 47), HRGD (SEQ ID NO: 51), GPYE (SEQ ID NO: 52), NHGG (SEQ ID NO: 53), THGG (SEQ ID NO: 54), GTHG (SEQ ID NO: 21), GSGS (SEQ ID NO: 56), GSGG (SEQ ID NO: 57), GGGG (SEQ ID NO: 58), GRGG (SEQ ID NO: 59), and GKGG (SEQ ID NO: 60). In some embodiments, a gene-editing protein comprises a C-terminal GTHG produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C.

[0361] In some cases, a cell is contacted with a demethylating agent during the process of gene-editing. In embodiments, the demethylating agent is selected from 5-azacitidine and 5-aza-2′-deoxycitidine (decitabine).

[0362] In some embodiments, the gene-editing protein comprises: (a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13; and (b) the nuclease domain comprising a catalytic domain, the catalytic domain comprising a hybrid of the catalytic domains of Fokl and Stsl, comprising the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Fokl with at least one of the domains of Fokl being substituted in whole or in part with the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Stsl and optionally comprising at least one mutation.

[0363] In various embodiments, the cell is transfected (e.g., contacted) with a synthetic nucleic acid encoding the gene-editing protein at about 30° C. to about 35° C., e.g., without limitation about 33° C. In embodiments, the contacting occurs at about 30° C. In some embodiments, the contacting occurs at about 31° C. In some embodiments, the contacting occurs at about 32° C. In some embodiments, the contacting occurs at about 33° C. In some embodiments, the contacting occurs at about 34° C. In some embodiments, the contacting occurs at about 35° C. In embodiments, the gene-editing protein is functionally temperature-switchable. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. to about 35° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 31° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 32° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 33° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 34° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 35° C.

[0364] In embodiments, the gene-edited cell, as disclosed herein, is also reprogrammed, as disclosed herein. In some embodiments, the gene-editing is contemporaneously with reprogramming. In various embodiments, the gene-editing is before the reprogramming. In other embodiments, the gene-editing is after the reprogramming. In cases when a cell is reprogramming, the cell may be a differentiated or a non-pluripotent cell. In some cases, the differentiated or a non-pluripotent cell is a skin cell (e.g., a fibroblast or a keratinocyte).Resveratrol

[0365] In yet another aspect, the present disclosure relates to use of Resveratrol treatment prior to transfection, e.g., with a synthetic nucleic acid encoding a gene-editing protein, and / or after transfection, e.g., with a synthetic nucleic acid encoding a gene-editing protein.

[0366] Gene editing technology, which enables the precision modification of DNA in living cells, is being developed for the treatment of various diseases, including genetic diseases and cancer. Gene editing commonly employs sequence-specific endonucleases to create double strand breaks in genomic DNA and relies on the cell's DNA repair mechanisms to apply the desired changes. Precise sequence modifications, such as single-base changes, rely on the homology directed repair (HDR) mechanism. Despite its essential role in gene repair, HDR occurs at a very low frequency in many cells compared to other repair mechanisms. The present disclosure discloses the impact of resveratrol, a small molecule extracted from grape skin, that promotes the expression of key HDR factors and induces cell cycle arrest at S phase in porcine fetal fibroblasts, on single-base editing efficiency in primary human fibroblasts. Following treatment with resveratrol, fibroblasts was co-transfected with mRNA encoding a chromatin context-sensitive gene-editing protein targeting the AAVS1 safe-harbor locus, and a single-stranded DNA repair template designed to introduce a SfoI restriction-enzyme site through a G-to-C mutation. Single-base editing efficiency was determined by restriction fragment length polymorphism (RFLP) analysis. Resveratrol treatment prior to transfection increased the S and G2-phase population 2.3-fold and increased HDR efficiency 2-fold compared to untreated cells. Application of resveratrol after transfection (i.e., no cell cycle synchronization) yielded further improvement in single-base editing efficiency (>2-fold), suggesting that the effects of resveratrol on HDR are not confined to cell-cycle control. This disclosure provides proof of concept that Resveratrol treatment provides a straightforward method for improving HDR efficiency in primary human fibroblasts and serves as a useful tool in the development of HDR-based gene-editing therapies.

[0367] The process of contacting Resveratrol with a cell in advance of gene editing is illustrated in FIG. 58. As shown in FIG. 58, Resveratrol arrests the cell in S or G2 phase and enhances the efficiency of subsequent gene-editing.

[0368] Notably, Resveratrol treatment before transfecting with a gene-editing machinery (e.g., a synthetic nucleic acid encoding a gene-editing protein) appears to arrest the majority of cells in S / G2; this pre-treatment enhances the efficiency of subsequent gene-editing. Also, Resveratrol treatment after transfecting with a gene-editing machinery (e.g., a synthetic nucleic acid encoding a gene-editing protein) enhances the efficiency of ongoing gene-editing. Therefore, in embodiments, cells may be pre-treated with Resveratrol and / or post-treated with Resveratrol.

[0369] Any of the herein-disclosed gene-editing methods may comprise pre-treatment with Resveratrol and / or post-treated with Resveratrol.Reprogramming Methods

[0370] In embodiments, the present disclosure relates to RNA-based modifications, e.g., reprogramming and / or gene-editing. In some embodiments, an RNA molecule encodes a gene-editing protein. In some embodiments, an RNA molecule encodes a reprogramming factor.

[0371] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides.

[0372] In various embodiments, the present invention relates to the reprogramming of iPSCs to Monocytes, which can then be further differentiated into M1 and / or M2 macrophages, using non-viral, RNA-based means. iPSCs, namely pluripotent or less differentiated cells, can be reprogrammed from non-pluripotent or differentiated cells, including fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0373] In some embodiments, the method for reprogramming a non-pluripotent cell comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and wherein step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0374] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed to a less differentiated state; and wherein step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0375] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors; and wherein step (c) is performed at least twice and the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections to result in the cell being reprogrammed to a less differentiated state and occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0376] In some embodiments, the method for reprogramming a non-pluripotent cell, comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and wherein step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell.

[0377] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed to a less differentiated state; and wherein step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell to a less differentiated state.

[0378] In some embodiments, the method for reprogramming a non-pluripotent cell, comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; (c) transfecting the non-pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (d) repeating step (c) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the non-pluripotent cell being reprogrammed, wherein steps (c) and (d) occur in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

[0379] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (d) repeating step (c) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state, wherein steps (c) and (d) occur in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0380] In some embodiments, the method for reprogramming a non-pluripotent cell comprises: (a) providing a non-pluripotent cell, the non-pluripotent cell being derived from a biopsy of a human subject; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with a synthetic RNA molecule, wherein: the synthetic RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the non-pluripotent cell expressing the one or more reprogramming factor(s) which reprograms the non-pluripotent cell; and step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

[0381] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) providing a non-pluripotent cell; (b) culturing the cell; and (c) transfecting the cell with a synthetic RNA molecule, wherein: the RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state, and step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell to a less differentiated state.

[0382] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) providing a non-pluripotent cell; (b) culturing the cell in a medium containing ingredients that support reprogramming of the cell to a less differentiated state; and (c) transfecting the cell with a synthetic RNA molecule, wherein: the RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state, and step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a feeder cell conditioned medium.

[0383] In some embodiments, the method for reprogramming a cell to a less differentiated state comprises: (a) providing a non-pluripotent cell; (b) culturing the cell in a medium containing albumin and ingredients that support reprogramming of the cell to a less differentiated state, wherein the albumin is treated with an ion-exchange resin or charcoal; (c) transfecting the cell with a synthetic RNA molecule, wherein the RNA molecule encoding one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, wherein the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state.

[0384] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) culturing a differentiated cell with a reprogramming medium; (b) transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (c) repeating step (b) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state, wherein steps (a)-(c) are performed without using feeder cells and occur in the presence of a feeder cell conditioned medium.

[0385] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin is treated with an ion-exchange resin or charcoal; b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and c. repeating step (b) at least twice during 5 consecutive days to result in the cell being reprogrammed to a less differentiated state.

[0386] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin is treated with sodium octanoate; brought to a temperature of at least about 40° C.; and treated with an ion-exchange resin or charcoal; b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding one or more reprogramming transcription factors and wherein the transfecting results in the cell expressing the one or more synthetic RNA molecules; and c. repeating step (b) at least twice during about 5 consecutive days to result in the cell being reprogrammed to a less differentiated state. In embodiments, the reprogramming is non-viral. In embodiments, the reprogramming factor is one or more of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0387] In some embodiments, iPSCs are obtained and Monocytes, which can then be further differentiated into M1 and / or M2 macrophages, are generated via cell reprogramming with non-immunogenic messenger RNA (mRNA) encoding one or more reprogramming factors in a defined, animal component-free process. In some embodiments the process is immunosuppressant-free. In some embodiments, the process is animal component-free. In some embodiments, the process is defined. In some embodiments, iPSCs are generated from adult human dermal fibroblasts using a high-efficiency, immunosuppressant-free mRNA-based protocol, whereupon iPSCs are differentiated into monocytes using a 28-day monolayer protocol. Beginning on day 14, cells can be harvested every 3-4 days. CD14+ isolation yielded >95% CD14+ cells with an average yield of 4.1×104 cells per cm2 per harvest. iPSC-derived monocytes were compared to peripheral blood mononuclear cell (PBMC)-derived monocytes for expression of key hematopoietic and myeloid-lineage markers CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα. In some embodiments, the differentiated monocytes are characterized by downregulation of Nanog and Oct4 and / or changes in expression of CD11b, CD14, CD33, CD45, CD80, CD163, CD206, and SIRPα, e.g., relative to the source cells. In some embodiments, rtPCR analysis is used to characterize monocytes. In some embodiments, monocytes are further differentiated into M1 and / or M2 macrophages. iPSC-derived monocytes can be further differentiated into macrophages by exposure to MCSF for 3-4 days. The macrophages can be assessed for their ability to polarize, secrete pro- and anti-inflammatory cytokines, and for cytotoxic activity when co-cultured with cancer cells. M1 macrophages can be polarized with interferon gamma (IFN-γ, 50 ng / mL) and lipopolysaccharide (LPS, 10 ng / mL) for 48 hours, whereas M2 macrophages can be treated with IL-4 (10 ng / mL) for 48 hours. The efficiency of iPSC-derived monocytes differentiation into macrophages can be assessed by cell adherence, morphology, and surface marker expression (CD14, CD45, CD163). The ability of M1 and M2 polarized iPSC-derived macrophages to secrete TNFα, IL-12p70, and IL-10 can be assayed and compared to PBMC-derived macrophages. Finally, the ability of M1 and M2 polarized iPSC-derived macrophages to kill cancer cells can be assayed.

[0388] Cells can be reprogrammed by exposing them to specific extracellular cues and / or by ectopic expression of specific proteins, microRNAs, etc. While several reprogramming methods have been previously described, most that rely on ectopic expression require the introduction of exogenous DNA, which can carry mutation risks. DNA-free reprogramming methods based on direct delivery of reprogramming proteins have been reported. However, these methods are too inefficient and unreliable for commercial use. In addition, RNA-based reprogramming methods have been described (see, e.g., Angel. MIT Thesis. 2008. 1-56; Angel et al. PLoS ONE. 2010. 5, 107; Warren et al. Cell Stem Cell. 2010. 7, 618-630; Angel. MIT Thesis. 2011. 1-89; and Lee et al., Cell. 2012. 151, 547-558; the contents of all of which are hereby incorporated by reference). However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require many transfections (resulting in significant expense and opportunity for error), can reprogram only a limited number of cell types, can reprogram cells to only a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeders. The many drawbacks of previously disclosed RNA-based reprogramming methods make them undesirable for research, therapeutic or cosmetic use.

[0389] In some embodiments, reprogramming is performed by transfecting cells with one or more nucleic acids encoding one or more reprogramming factors, including, but not limited to Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In one embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a pluripotent stem cell. In another embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a glucose-responsive insulin-producing cell. Examples of other cells that can be reprogrammed and other cells to which a cell can be reprogrammed include, but are not limited to skin cells, pluripotent stem cells, MSCs, mesenchymal stromal / stem cells, β-cells, retinal pigmented epithelial cells, hematopoietic cells, hematopoietic stem cells, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells. In one embodiment, the cell is contacted with a medium that supports the reprogrammed cell. In one embodiment, the medium also supports the cell.

[0390] Importantly, infecting skin cells with viruses encoding Oct4, Sox2, Klf4, and c-Myc, combined with culturing the cells in a medium that supports the growth of cardiomyocytes, has been reported to cause reprogramming of the skin cells to cardiomyocytes, without first reprogramming the skin cells to pluripotent stem cells (See Efs et al Nat Cell Biol. 2011; 13:215-22, the contents of which are hereby incorporated by reference). In certain situations, direct reprogramming (reprogramming one somatic cell to another somatic cell without first reprogramming the somatic cell to a pluripotent stem cell, also known as “transdifferentiation”) may be desirable, in part because culturing pluripotent stem cells can be time-consuming and expensive, the additional handling involved in establishing and characterizing a stable pluripotent stem cell line can carry an increased risk of contamination, and the additional time in culture associated with first producing pluripotent stem cells can carry an increased risk of genomic instability and the acquisition of mutations, including point mutations, copy-number variations, and karyotypic abnormalities.

[0391] In embodiments, fewer total transfections may be required to reprogram a cell according to the methods of the present invention than according to other methods. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein from about 1 to about 12 transfections are performed during about 20 consecutive days, or from about 4 to about 10 transfections are performed during about 15 consecutive days, or from about 4 to about 8 transfections are performed during about 10 consecutive days. It is recognized that when a cell is contacted with a medium containing nucleic acid molecules, the cell may likely come into contact with and / or internalize more than one nucleic acid molecule either simultaneously or at different times. A cell can therefore be contacted with a nucleic acid more than once, e.g., repeatedly, even when a cell is contacted only once with a medium containing nucleic acids.

[0392] Of note, nucleic acids can contain one or more non-canonical or “modified” residues as described herein. For instance, any of the non-canonical nucleotides described herein can be used in the present reprogramming methods. In one embodiment, pseudouridine-5′-triphosphate can be substituted for uridine-5′-triphosphate in an in vitro-transcription reaction to yield synthetic RNA, wherein up to 100% of the uridine residues of the synthetic RNA may be replaced with pseudouridine residues. In vitro-transcription can yield RNA with residual immunogenicity, even when pseudouridine and 5-methylcytidine are completely substituted for uridine and cytidine, respectively (see, e.g., Angel. Reprogramming Human Somatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge, MA: MIT; 2011, the contents of which are hereby incorporated by reference). For this reason, it is common to add an immunosuppressant to the transfection medium when transfecting cells with RNA. In certain situations, adding an immunosuppressant to the transfection medium may not be desirable, in part because the recombinant immunosuppressant most commonly used for this purpose, B18R, can be expensive and difficult to manufacture. In one embodiment, the immunosuppressant is B18R or a biologically active fragment, analogue, variant or family-member thereof or dexamethasone or a derivative thereof. In one embodiment, the transfection medium does not contain an immunosuppressant, and the nucleic-acid dose is chosen to prevent excessive toxicity. In another embodiment, the nucleic-acid dose is less than about 1 mg / cm2 of tissue or less than about 1 mg / 100,000 cells or less than about 10 mg / kg.

[0393] In various cases, transfection of a cell with synthetic nucleic acids for reprogramming the cell may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV):where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In any of the herein-disclosed aspects or embodiments, a synthetic RNA molecule may be in the form of a circular RNA (circRNA). The circRNA are manufactured by methods do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA, the circRNA are manufactured by methods that do not require ribozymes to yield a circRNA, and / or the circRNA are manufactured by methods that do not require HPLC-based purification, e.g., post-ligation. A nucleic acid that can be manufactured into a circRNA has the structure: 5′-X-Y-A-IRES-B-CDS-C-Y′-Z 3′. Here, Y and Y′ each independently comprise one or more nucleotides and Y and Y′ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null. The CDS of a circRNA may encode one or more proteins of interest, the protein of interest being one or more reprogramming factors, optionally selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, Aicda, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof. In some cases, the CDS encodes two, three, four, five, six, seven, eight, nine, ten, eleven, or more reprogramming factor(s). Additional details regarding circRNAs useful in the present disclosure are described in PCT / US2022 / 026564, the contents of which are incorporated herein by reference in its entirety.

[0395] Reprogrammed cells produced according to certain embodiments of the present invention are suitable for therapeutic and / or cosmetic applications as they do not contain undesirable exogenous DNA sequences, and they are not exposed to animal-derived or human-derived products, which may be undefined, and which may contain toxic and / or pathogenic contaminants. Furthermore, the high speed, efficiency, and reliability of certain embodiments of the present invention may reduce the risk of acquisition and accumulation of mutations and other chromosomal abnormalities. Certain embodiments of the present invention can thus be used to generate cells that have a safety profile adequate for use in therapeutic and / or cosmetic applications. For example, reprogramming cells using RNA and the medium of the present invention, wherein the medium does not contain animal or human-derived components, can yield cells that have not been exposed to allogeneic material. Certain embodiments are therefore directed to a reprogrammed cell that has a desirable safety profile. In one embodiment, the reprogrammed cell has a normal karyotype. In another embodiment, the reprogrammed cell has fewer than about 5 copy-number variations (CNVs) relative to the patient genome, such as fewer than about 3 copy-number variations relative to the patient genome, or no copy-number variations relative to the patient genome. In yet another embodiment, the reprogrammed cell has a normal karyotype and fewer than about 100 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 50 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 10 single nucleotide variants in coding regions relative to the patient genome.

[0396] Endotoxins and nucleases can co-purify and / or become associated with other proteins, such as serum albumin. Recombinant proteins, in particular, can often have high levels of associated endotoxins and nucleases, due in part to the lysis of cells that can take place during their production. Endotoxins and nucleases can be reduced, removed, replaced or otherwise inactivated by many of the methods of the present invention, including, for example, by acetylation, by addition of a stabilizer such as sodium octanoate, followed by heat treatment, by the addition of nuclease inhibitors to the albumin solution and / or medium, by crystallization, by contacting with one or more ion-exchange resins, by contacting with charcoal, by preparative electrophoresis or by affinity chromatography. In embodiments, partially or completely reducing, removing, replacing, or otherwise inactivating endotoxins and / or nucleases from a medium and / or from one or more components of a medium is provided and this can increase the efficiency with which cells can be transfected and reprogrammed. Certain embodiments are therefore directed to a method for transfecting a cell with one or more nucleic acids, wherein the transfection medium is treated to partially or completely reduce, remove, replace or otherwise inactivate one or more endotoxins and / or nucleases. Other embodiments are directed to a medium that causes minimal degradation of nucleic acids. In one embodiment, the medium contains less than about 1 EU / mL, or less than about 0.1 EU / mL, or less than about 0.01 EU / mL.

[0397] In certain situations, protein-based lipid carriers such as serum albumin can be replaced with non-protein-based lipid carriers such as methyl-beta-cyclodextrin. The medium of the present invention can also be used without a lipid carrier, for example, when transfection is performed using a method that may not require or may not benefit from the presence of a lipid carrier, for example, using one or more lipid-based transfection reagents, polymer-based transfection reagents or peptide-based transfection reagents or using electroporation. Many protein-associated molecules, such as metals, can be highly toxic to cells in vivo. This toxicity can cause decreased viability, as well as the acquisition of mutations. Certain embodiments thus have the additional benefit of producing cells that are free from toxic molecules.

[0398] The associated-molecule component of a protein can be measured by suspending the protein in solution and measuring the conductivity of the solution. Certain embodiments are therefore directed to a medium that contains a protein, wherein about a 10% solution of the protein in water has a conductivity of less than about 500 μmho / cm. In one embodiment, the solution has a conductivity of less than about 50 μmho / cm. In another embodiment, less than about 0.65% of the dry weight of the protein comprises lipids and / or less than about 0.35% of the dry weight of the protein comprises free fatty acids.

[0399] Certain embodiments are therefore directed to a method for transfecting a cell with a nucleic acid, wherein the cell is transfected more than once, and wherein the amount of nucleic acid delivered to the cell is different for two of the transfections. In one embodiment, the cell proliferates between two of the transfections, and the amount of nucleic acid delivered to the cell is greater for the second of the two transfections than for the first of the two transfections. In another embodiment, the cell is transfected more than twice, and the amount of nucleic acid delivered to the cell is greater for the second of three transfections than for the first of the same three transfections, and the amount of nucleic acid delivered to the cells is greater for the third of the same three transfections than for the second of the same three transfections. In yet another embodiment, the cell is transfected more than once, and the maximum amount of nucleic acid delivered to the cell during each transfection is sufficiently low to yield at least about 80% viability for at least two consecutive transfections.

[0400] In embodiments, there are provided methods in which modulating the amount of nucleic acid delivered to a population of proliferating cells in a series of transfections can result in both an increased effect of the nucleic acid and increased viability of the cells. In embodiments, when cells are contacted with one or more nucleic acids encoding one or more reprogramming factors in a series of transfections, the efficiency of reprogramming can be increased when the amount of nucleic acid delivered in later transfections is greater than the amount of nucleic acid delivered in earlier transfections, for at least part of the series of transfections. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein one or more nucleic acids is repeatedly delivered to the cell in a series of transfections, and the amount of the nucleic acid delivered to the cell is greater for at least one later transfection than for at least one earlier transfection. In one embodiment, the cell is transfected from about 2 to about 10 times, or from about 3 to about 8 times, or from about 4 to about 6 times. In another embodiment, the one or more nucleic acids includes at least one RNA molecule, the cell is transfected from about 2 to about 10 times, and the amount of nucleic acid delivered to the cell in each transfection is the same as or greater than the amount of nucleic acid delivered to the cell in the most recent previous transfection. In yet another embodiment, the amount of nucleic acid delivered to the cell in the first transfection is from about 20 ng / cm2 to about 250 ng / cm2, or from 100 ng / cm2 to 600 ng / cm2. In yet another embodiment, the cell is transfected about 5 times at intervals of from about 12 to about 48 hours, and the amount of nucleic acid delivered to the cell is about 25 ng / cm2 for the first transfection, about 50 ng / cm2 for the second transfection, about 100 ng / cm2 for the third transfection, about 200 ng / cm2 for the fourth transfection, and about 400 ng / cm2 for the fifth transfection. In yet another embodiment, the cell is further transfected at least once after the fifth transfection, and the amount of nucleic acid delivered to the cell is about 400 ng / cm2.

[0401] Several molecules can be added to media by conditioning. Certain embodiments are therefore directed to a medium that is supplemented with one or more molecules that are present in a conditioned medium. In one embodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In another embodiment, the medium is supplemented with TGF-β or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, a cell is reprogrammed according to the method of the present invention, wherein the medium is not supplemented with TGF-β for from about 1 to about 5 days and is then supplemented with TGF-β for at least about 2 days. In yet another embodiment, the medium is supplemented with IL-6, IL-6R or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, the medium is supplemented with a sphingolipid or a fatty acid. In still another embodiment, the sphingolipid is lysophosphatidic acid, lysosphingomyelin, sphingosine-1-phosphate or a biologically active analogue, variant or derivative thereof.

[0402] In addition to mitotically inactivating cells, under certain conditions, irradiation can change the gene expression of cells, causing cells to produce less of certain proteins and more of certain other proteins than non-irradiated cells, for example, members of the Wnt family of proteins. In addition, certain members of the Wnt family of proteins can promote the growth and transformation of cells. In embodiments, the efficiency of reprogramming can be greatly increased by contacting a cell with a medium that is conditioned using irradiated feeders instead of mitomycin-c-treated feeders. In embodiments, the increase in reprogramming efficiency observed when using irradiated feeders is caused in part by Wnt proteins that are secreted by the feeders. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein the cell is contacted with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, family-member or agonist thereof, including agonists of downstream targets of Wnt proteins, and / or agents that mimic one or more of the biological effects of Wnt proteins, for example, 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

[0403] Because of the low efficiency of many DNA-based reprogramming methods, these methods may be difficult or impossible to use with cells derived from patient samples, which may contain only a small number of cells. In contrast, the high efficiency of certain embodiments of the present invention can allow reliable reprogramming of a small number of cells, including single cells. Certain embodiments are directed to a method for reprogramming a small number of cells. Other embodiments are directed to a method for reprogramming a single cell. In one embodiment, the cell is contacted with one or more enzymes. In another embodiment, the enzyme is collagenase. In yet another embodiment, the collagenase is animal-component free. In one embodiment, the collagenase is present at a concentration of from about 0.1 mg / mL to about 10 mg / mL, or from about 0.5 mg / mL to about 5 mg / mL. In another embodiment, the cell is a blood cell. In yet another embodiment, the cell is contacted with a medium containing one or more proteins that is derived from the patient's blood. In still another embodiment, the cell is contacted with a medium comprising: DMEM / F12+2 mM L-alanyl-L-glutamine+from about 5% to about 25% patient-derived serum, or from about 10% to about 20% patient-derived serum, or about 20% patient-derived serum.

[0404] In embodiments, transfecting cells with a mixture of RNA encoding Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof using the medium of the present invention can cause the rate of proliferation of the cells to increase. When the amount of RNA delivered to the cells is too low to ensure that all of the cells are transfected, only a fraction of the cells may show an increased proliferation rate. In certain situations, such as when generating a personalized therapeutic, increasing the proliferation rate of cells may be desirable, in part because doing so can reduce the time necessary to generate the therapeutic, and therefore can reduce the cost of the therapeutic. Certain embodiments are therefore directed to a method for transfecting a cell with a mixture of RNA encoding Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In one embodiment, the cell exhibits an increased proliferation rate. In another embodiment, the cell is reprogrammed.

[0405] While detailed examples are provided herein for the production of specific types of cells and for the production of therapeutics comprising specific types of cells, it is recognized that the methods of the present invention can be used to produce many other types of cells, and to produce therapeutics comprising one or more of many other types of cells, for example, by reprogramming a cell according to the methods of the present invention, and culturing the cell under conditions that mimic one or more aspects of development by providing conditions that resemble the conditions present in the cellular microenvironment during development.

[0406] Other embodiments are directed to a method for reprogramming a cell. In one embodiment, the cell is reprogrammed by contacting the cell with one or more nucleic acids. In one embodiment, the cell is contacted with a plurality of nucleic acids encoding at least one of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In another embodiment, the cell is contacted with a plurality of nucleic acids encoding a plurality of proteins including: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0407] Illustrative subjects or patients refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0408] In some embodiments, a synthetic RNA molecule is used to reprogram iPSCs into monocytes, which can then be further differentiated into M1 and / or M2 macrophages. In embodiments, the synthetic RNA molecule is mRNA. In embodiments, the synthetic RNA molecule is in vitro transcribed. In embodiments, the synthetic RNA is a circRNA.

[0409] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides. In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein, and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0410] In some embodiments, the synthetic RNA comprises a 5′ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a sequence that increases RNA stability in vivo, and the 5′-UTR optionally comprises an alpha-globin or beta-globin 5′-UTR. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a sequence that increases RNA stability in vivo, and the 3′-UTR optionally comprises an alpha-globin or beta-globin 3′-UTR. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0411] Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′-UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20 nt and about 250 nt or between about 120 nt and about 150 nt long. In a further embodiment, the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

[0412] In some embodiments, the RNA comprises a tail composed of a plurality of adenines with one or more guanines.

[0413] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0414] In embodiments, the one or more other nucleotides comprises deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0415] In embodiments, the one or more other nucleotides comprises deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0416] In embodiments, the one or more other nucleotides comprises deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0417] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues and deoxycytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region comprises fewer than 50% deoxyadenosine residues.

[0418] In embodiments, the one or more other nucleotides comprises guanosine residues.

[0419] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues. In embodiments, the tail region comprises more than 50% guanosine residues.

[0420] In embodiments, the one or more other nucleotides comprises cytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0421] In embodiments, the one or more other nucleotides comprises uridine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0422] In embodiments, the one or more other nucleotides comprise guanosine residues and cytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0423] In embodiments, the tail region comprises fewer than 50% adenosine residues.

[0424] In embodiments, the tail is (A)150 (SEQ ID NO: 61). In embodiments, the tail is (A39G)3(A)30 (SEQ ID NO: 62). In embodiments, the tail is (A19G)7(A)10 (SEQ ID NO: 63). In embodiments, the tail is (A9G)15 (SEQ ID NO: 64).

[0425] In embodiments, the length of the tail region is between about 80 nucleotides and about 120 nucleotides, about 120 nucleotides and about 160 nucleotides, about 160 nucleotides and about 200 nucleotides, about 200 nucleotides and about 240 nucleotides, about 240 nucleotides and about 280 nucleotides, or about 280 nucleotides and about 320 nucleotides.

[0426] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0427] In embodiments, the RNA comprises a 5′ cap structure. In embodiments, the RNA 5′-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5′-UTR comprises a sequence that increases RNA stability in vivo, and the 5′-UTR may comprise an alpha-globin or beta-globin 5′-UTR.

[0428] In embodiments, the RNA 3′-UTR comprises a sequence that increases RNA stability in vivo, and the 3′-UTR may comprise an alpha-globin or beta-globin 3′-UTR. In embodiments, the RNA comprises a 3′ poly(A) tail. In embodiments, the RNA 3′ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0429] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0430] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is synthetic.

[0431] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides.

[0432] In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0433] Further description of reprogramming is found in one or more of WO / 2013 / 086008, WO / 2014 / 071219, WO / 2015 / 117021, WO / 2016 / 131052, WO / 2018 / 035377, WO / 2019 / 191341, WO / 2021 / 003462, WO2021 / 231549, or WO2021 / 222389. The entire contents of which are incorporated by reference in their entirety.Gene-Editing

[0434] In any herein disclosed aspect or embodiment, a cell is gene-edited.

[0435] In embodiments, gene-editing a cell comprises contacting the cell with a synthetic nucleic acid encoding one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR / Cas9, Cas9, xCas9, Cas12a(Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyz (SEQ ID NO: 16), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.

[0436] In embodiments, the gene-editing protein comprises: (i) a DNA-binding domain comprising a plurality of repeat sequences and (ii) the nuclease domain comprising a catalytic domain of a nuclease. In embodiments, the at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 22) and is optionally between 36 and 39 amino acids long, where:

[0437] v is Q, D or E,

[0438] w is S or N,

[0439] x is I, H, N, or I,

[0440] y is D, A, I, N, H, K, S, G, or null,

[0441] z is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), and

[0442] α is four consecutive amino acids.

[0443] In embodiments, α comprises at least one glycine (G) residue. In embodiments, α comprises at least one histidine (H) residue. In embodiments, α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35. In embodiments, α comprises at least one aspartic acid (D) residue. In embodiments, α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.

[0444] In embodiments, α comprises one or more hydrophilic residues, optionally selected from: a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K); a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C); a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).

[0445] In some embodiments, α comprises one or more polar and positively charged hydrophilic amino acids selected from arginine (R) and lysine (K). In some embodiments, α comprises one or more polar and neutral of charge hydrophilic amino acids selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In some embodiments, α comprises one or more polar and negatively charged hydrophilic amino acids selected from aspartate (D) and glutamate (E). In some embodiments, α comprises one or more aromatic, polar and positively charged hydrophilic amino acids selected from histidine (H).

[0446] In embodiments, α comprises one or more hydrophobic residues, optionally selected from: a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In some embodiments, α comprises one or more hydrophobic, aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In some embodiments, α comprises one or more aromatic amino acids selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In embodiments, the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.

[0447] In embodiments, α is selected from GHGG (SEQ ID NO: 31), HGSG (SEQ ID NO: 32), HGGG (SEQ ID NO: 33), from GGHD (SEQ ID NO: 34), GAHD (SEQ ID NO: 35), AHDG (SEQ ID NO: 36), PHDG (SEQ ID NO: 37), GPHD (SEQ ID NO: 38), GHGP (SEQ ID NO: 39), PHGG (SEQ ID NO: 40), PHGP (SEQ ID NO: 41), AHGA (SEQ ID NO: 42), LHGA (SEQ ID NO: 43), VHGA (SEQ ID NO: 44), IVHG (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), RHGD (SEQ ID NO: 47), RDHG (SEQ ID NO: 48), RHGE (SEQ ID NO: 49), HRGE (SEQ ID NO: 50), RHGD (SEQ ID NO: 47), HRGD (SEQ ID NO: 51), GPYE (SEQ ID NO: 52), NHGG (SEQ ID NO: 53), THGG (SEQ ID NO: 54), GTHG (SEQ ID NO: 21), GSGS (SEQ ID NO: 56), GSGG (SEQ ID NO: 57), GGGG (SEQ ID NO: 58), GRGG (SEQ ID NO: 59), and GKGG (SEQ ID NO: 4260

[0448] In embodiments, the gene-editing protein has a DNA binding domain having at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (SEQ ID NO: 65; the “*RVD*” corresponds to the dinucleotide “xy” of SEQ ID NO: 22).

[0449] In embodiments, the repeat sequence is 33 or 34 amino acids long. In embodiments, the repeat sequence is 36-39 amino acids long. In some embodiments, the repeat sequence is 36 amino acids long. In some embodiments, the repeat sequence is 37 amino acids long. In some embodiments, the repeat sequence is 38 amino acids long. In some embodiments, the repeat sequence is 39 amino acids long.

[0450] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17) or LTPvQVVAIAwxyzGTHG (SEQ ID NO: 18) and is from 36 to 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20) and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In some embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C.

[0451] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 22) and is from 36 to 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), a is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, a is selected from GHGG (SEQ ID NO: 31), HGSG (SEQ ID NO: 32), HGGG (SEQ ID NO: 33), GGHD (SEQ ID NO: 34), GAHD (SEQ ID NO: 35), AHDG (SEQ ID NO: 36), PHDG (SEQ ID NO: 37), GPHD (SEQ ID NO: 38), GHGP (SEQ ID NO: 39), PHGG (SEQ ID NO: 40), PHGP (SEQ ID NO: 41), AHGA (SEQ ID NO: 42), LHGA (SEQ ID NO: 43), VHGA (SEQ ID NO: 44), IVHG (SEQ ID NO: 45), IHGM (SEQ ID NO: 46), RHGD (SEQ ID NO: 47), RDHG (SEQ ID NO: 48), RHGE (SEQ ID NO: 49), HRGE (SEQ ID NO: 50), RHGD (SEQ ID NO: 47), HRGD (SEQ ID NO: 51), GPYE (SEQ ID NO: 52), NHGG (SEQ ID NO: 53), THGG (SEQ ID NO: 54), GTHG (SEQ ID NO: 21), GSGS (SEQ ID NO: 56), GSGG (SEQ ID NO: 57), GGGG (SEQ ID NO: 58), GRGG (SEQ ID NO: 59), and GKGG (SEQ ID NO: 60). In some embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 33° C. GTHG (SEQ ID NO: 21). In various embodiments, a gene-editing protein comprises a C-terminal GTHG (SEQ ID NO: 21) produces more efficient editing at the target locus than TALENs at 37° C. Certain embodiments are directed to a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising a first region that recognizes a predetermined nucleotide sequence and a second region with endonuclease activity, wherein the first region contains an artificial TAL effector repeat domain comprising one or more repeat units about 36 amino acids in length which differ from each other by no more than seven amino acids, and wherein the repeat domain is engineered for recognition of the predetermined nucleotide sequence. In one embodiment, the first region contains the amino acid sequence: LTPXQVVAIAS (SEQ ID NO: 29) where X can be either E or Q. In another embodiment, the amino acid sequence LTPXQVVAIAS (SEQ ID NO: 29) of the encoded non-naturally occurring fusion protein is immediately followed by an amino acid sequence selected from: HD, NG, NS, NI, NN, and N. In a further embodiment, the fusion protein comprises restriction endonuclease activity.

[0452] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzHG, (SEQ ID NO: 30) wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG, (SEQ ID NO: 30) wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG, (SEQ ID NO: 30) wherein “v” is D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAIwyzHG (SEQ ID NO: 55), wherein “v” is D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzHG (SEQ ID NO: 66), wherein “v” is D or E, “w” is S or N, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 30), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 23), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 30), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 25), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 26), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 27), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 28). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx (SEQ ID NO: 67), wherein “v” is D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy (SEQ ID NO: 16), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwyzGHGG (SEQ ID NO: 70), wherein “v” is Q, D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzGHGG, (SEQ ID NO: 71) wherein “v” is Q, D or E, “w” is S or N, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 17), wherein “v” is Q, D or E, V is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 23), GGKQALE (SEQ ID NO: 24), GGKQALETVQRLLPVLCQD (SEQ ID NO: 19), GGKQALETVQRLLPVLCQA (SEQ ID NO: 20), GKQALETVQRLLPVLCQD (SEQ ID NO: 69) or GKQALETVQRLLPVLCQA (SEQ ID NO: 68). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx (SEQ ID NO: 67), wherein “v” is Q, D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy (SEQ ID NO: 72), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G.

[0453] The above-mentioned gene-editing proteins comprise a repeat variable di-residue (RVD) at residue 12 or 13, e.g., at “x” and “y” in the various above-mentioned repeat sequences, e.g., LTPvQVVAIAwxyzα (SEQ ID NO: 22), which targets the DNA-binding domain to a target DNA molecule. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(null), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(null), and IG.

[0454] In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HD. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is N(null). In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HA. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is ND. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HI. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NN. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NH. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NK. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is HN. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NA. In some embodiments, the RVD recognizing an A residue in the nucleic acid molecule is Nl. In some embodiments, the RVD recognizing an A residue in the nucleic acid molecule is NS. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is NG. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is HG. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is H(null). In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is IG.

[0455] In some embodiments, alternative DNA binding domains are employed.

[0456] For example, the alternative DNA binding domains described herein are, in embodiments, paired with the novel engineered nuclease domains described herein.

[0457] For example, the alternative DNA binding domains described herein are, in embodiments, used in the conditional activity: temperature dependence methods described herein.

[0458] For example, the alternative DNA binding domains described herein are, in embodiments, used in the conditional activity: methylation status methods described herein.

[0459] In embodiments, the engineered gene-editing proteins do not require a thymine (T) in the zero position of the target site (“To”).

[0460] In embodiments, the engineered gene-editing proteins that comprise DNA-binding domains comprise alterations in the in the N-terminal region to remove the T0 requirement.

[0461] In embodiments, there is provided a method of gene-editing a cell with one or more of the present gene-editing proteins, optionally with also using a linear DNA repair template, optionally also using conditional activity methods described herein, where the target site lacks a thymine (T) in the zero position.

[0462] Wild type N-terminal region is characterized by the sequence: Asp225-IVGVGKQWSGARAL-Glu240 (DIVGVGKQWSGARALE; SEQ ID NO: 73). In embodiments, there is provided the engineered N-terminal region of Asp225-IVGVGKQKRGARAL-Glu240 (underling showing the change WS->KR) (DIVGVGKQKRGARALE; SEQ ID NO: 74).

[0463] In embodiments, there is provided an engineered N-terminal region in which KQWS is replaced with one or more amino acids, e.g., about 2-10 amino acids, or about 4-10 amino acids, or about 6-10 amino acids, or about 8-10 amino acids, or about 4 amino acids, or about 6 amino acids, or about 8 amino acids, or about 10 amino acids.

[0464] In embodiments, there is provided the engineered N-terminal region of Asp225-IVGVGGSKRGAGSGARAL-Glu244 (underling showing the change KQWS->GSKRGAGS) (DIVGVGGSKRGAGSGARALE; SEQ ID NO: 75).

[0465] In some cases, a cell is contacted with a demethylating agent during the process of gene-editing. In embodiments, the demethylating agent is selected from 5-azacitidine and 5-aza-2′-deoxycitidine (decitabine).

[0466] In some embodiments, the gene-editing protein comprises: (a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13; and (b) the nuclease domain comprising a catalytic domain, the catalytic domain comprising a hybrid of the catalytic domains of Fokl and Stsl, comprising the α1, α2, α3, α4, α5, α6, β1, β2, β, β4, β5, and β6 domains of Fokl with at least one of the domains of Fokl being substituted in whole or in part with the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Stsl and optionally comprising at least one mutation. In embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

[0467] In some embodiments, certain fragments of an endonuclease cleavage domain are used, including fragments that are truncated at the N-terminus, fragments that are truncated at the C-terminus, fragments that have internal deletions, and fragments that combine N-terminus, C-terminus, and / or internal deletions, which maintain part or all of the catalytic activity of the full endonuclease cleavage domain. Determining whether a fragment can maintain part, or all of the catalytic activity of the full domain can be accomplished by, for example, synthesizing a gene-editing protein that contains the fragment according to the methods of the present invention, inducing cells to express the gene-editing protein according to the methods of the present invention, and measuring the efficiency of gene editing. In some embodiments, a measurement of gene-editing efficiency is used to ascertain whether any specific fragment maintains part or all of the catalytic activity of the full endonuclease cleavage domain. Certain embodiments are therefore directed to a biologically active fragment of an endonuclease cleavage domain. In one embodiment, the endonuclease cleavage domain is selected from: FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineered variant or biologically active fragment thereof, or a hybrid or chimera thereof.

[0468] In embodiments, the gene-editing protein comprises a linker. In another embodiment, the linker connects a DNA-binding domain to a nuclease domain. In a further embodiment, the linker is between about 1 and about 10 amino acids long. In some embodiments, the linker is about 1, about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids long. In one embodiment, the gene-editing protein is capable of generating a nick or a double-strand break in a target DNA molecule.

[0469] In embodiments, the gene-editing protein is any of those described in International Patent Publication No. WO 2014 / 071219 or WO2021 / 231549, hereby incorporated by reference in their entireties.

[0470] In various embodiments, the cell is transfected (e.g., contacted) with a synthetic nucleic acid encoding the gene-editing protein at about 30° C. to about 35° C., e.g., without limitation about 33° C. In embodiments, the contacting occurs at about 30° C. In some embodiments, the contacting occurs at about 31° C. In some embodiments, the contacting occurs at about 32° C. In some embodiments, the contacting occurs at about 33° C. In some embodiments, the contacting occurs at about 34° C. In some embodiments, the contacting occurs at about 35° C. In embodiments, the gene-editing protein is functionally temperature-switchable. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. to about 35° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 30° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 31° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 32° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 33° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 34° C. In embodiments, the method further comprises the step of (c) culturing the contacted cell at about 35° C.

[0471] Further description of temperature-sensitive gene-editing is found in WO2021 / 231549. The entire contents of which are incorporated by reference in their entirety.

[0472] In embodiments, the synthetic nucleic acid encoding the gene-editing protein is transfected along with a repair template. In some cases, the repair template is a double stranded synthetic oligodeoxynucleotide (dsODNs). In embodiments, the dsODNs comprises a repair template and comprises the TTAGGG motif. In some cases, the dsODN comprises a repair template and lacks the TTAGGG motif and a separate dsODNs comprising the TTAGGG motif is transfected into the cell. In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides. In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein, and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0473] In some embodiments, the synthetic RNA comprises a 5′ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a sequence that increases RNA stability in vivo, and the 5′-UTR optionally comprises an alpha-globin or beta-globin 5′-UTR. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a sequence that increases RNA stability in vivo, and the 3′-UTR optionally comprises an alpha-globin or beta-globin 3′-UTR. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0474] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0475] In various cases, transfection of a cell with synthetic nucleic acids for gene-editing the cell may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (IV)where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or both of U.S. Pat. No. 10,501,404 and WO2021003462. The entire contents of which are incorporated by reference in their entirety.In any of the herein-disclosed aspects or embodiments, a synthetic RNA molecule encoding the gene-editing protein may be in the form of a circular RNA (circRNA). The circRNA are manufactured by methods do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA, the circRNA are manufactured by methods that do not require ribozymes to yield a circRNA, and / or the circRNA are manufactured by methods that do not require HPLC-based purification, e.g., post-ligation. A nucleic acid that can be manufactured into a circRNA has the structure: 5′-X-Y-A-IRES-B-CDS-C-Y′-Z 3′. Here, Y and Y′ each independently comprise one or more nucleotides and Y and Y′ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null. The CDS of a circRNA encodes the gene-editing protein(s). Additional details regarding circRNAs useful in the present disclosure are described in PCT / US2022 / 026564, the contents of which are incorporated herein by reference in its entirety.

[0477] In some embodiments, the synthetic RNA comprises a 5′ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a sequence that increases RNA stability in vivo, and the 5′-UTR optionally comprises an alpha-globin or beta-globin 5′-UTR. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a sequence that increases RNA stability in vivo, and the 3′-UTR optionally comprises an alpha-globin or beta-globin 3′-UTR. In some embodiments, the synthetic RNA comprises a 5′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3′ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0478] Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′-UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20 nt and about 250 nt or between about 120 nt and about 150 nt long. In a further embodiment, the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

[0479] In some embodiments, the RNA comprises a tail composed of a plurality of adenines with one or more guanines.

[0480] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0481] In embodiments, the one or more other nucleotides comprises deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0482] In embodiments, the one or more other nucleotides comprises deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0483] In embodiments, the one or more other nucleotides comprises deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0484] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues and deoxycytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region comprises fewer than 50% deoxyadenosine residues.

[0485] In embodiments, the one or more other nucleotides comprises guanosine residues.

[0486] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues. In embodiments, the tail region comprises more than 50% guanosine residues.

[0487] In embodiments, the one or more other nucleotides comprises cytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0488] In embodiments, the one or more other nucleotides comprises uridine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0489] In embodiments, the one or more other nucleotides comprise guanosine residues and cytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0490] In embodiments, the tail region comprises fewer than 50% adenosine residues.

[0491] In embodiments, the tail is (A)150 (SEQ ID NO: 61). In embodiments, the tail is (A39G)3(A)30 (SEQ ID NO: 62). In embodiments, the tail is (A19G)7(A)10 (SEQ ID NO: 63). In embodiments, the tail is (A9G)15 (SEQ ID NO: 64).

[0492] In embodiments, the length of the tail region is between about 80 nucleotides and about 120 nucleotides, about 120 nucleotides and about 160 nucleotides, about 160 nucleotides and about 200 nucleotides, about 200 nucleotides and about 240 nucleotides, about 240 nucleotides and about 280 nucleotides, or about 280 nucleotides and about 320 nucleotides.

[0493] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0494] In embodiments, the RNA comprises a 5′ cap structure. In embodiments, the RNA 5′-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5′-UTR comprises a sequence that increases RNA stability in vivo, and the 5′-UTR may comprise an alpha-globin or beta-globin 5′-UTR.

[0495] In embodiments, the RNA 3′-UTR comprises a sequence that increases RNA stability in vivo, and the 3′-UTR may comprise an alpha-globin or beta-globin 3′-UTR. In embodiments, the RNA comprises a 3′ poly(A) tail. In embodiments, the RNA 3′ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0496] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0497] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is synthetic. In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0498] Further description of gene-editing is found in one or more of WO / 2013 / 086008, WO / 2014 / 071219, WO / 2015 / 117021, WO / 2016 / 131052, WO / 2018 / 035377, WO / 2019 / 191341, WO / 2021 / 003462, WO2021 / 231549, or WO2021 / 222389. The entire contents of which are incorporated by reference in their entirety.RNA Modifications

[0499] In embodiments, the present disclosure relates to RNA-based modifications, e.g., reprogramming and / or gene-editing. In some embodiments, an RNA molecule encodes a gene-editing protein. In some embodiments, a RNA molecule encodes a reprogramming factor.

[0500] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides.

[0501] In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein, and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides.

[0502] In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0503] In embodiments, the synthetic RNA molecule is mRNA comprising one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0504] In some embodiments, the one or more non-canonical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine. In some embodiments, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the non-canonical nucleotides are one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0505] In some embodiments, at least about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of cytidine residues are non-canonical nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine.

[0506] In some embodiments, at least about 20%, or about 30%, or about 40%, or about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of uridine residues are non-canonical nucleotides selected from 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0507] In some embodiments, at least about 10% (e.g., 10%, or about 20%, or about 30%, or about 40%, or about 50%) of guanosine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the RNA contains no more than about 50% 7-deazaguanosine in place of guanosine residues.

[0508] In some embodiments, the synthetic RNA molecule does not contain non-canonical nucleotides in place of adenosine residues.

[0509] Other non-canonical nucleotides that can be used in place of or in combination with 5-methyluridine include but are not limited to: pseudouridine and 5-methylpseudouridine (a.k.a. “1-methylpseudouridine”, a.k.a. “N1-methylpseudouridine”) or one or more derivatives thereof. Other non-canonical nucleotides that can be used in place of or in combination with 5-methylcytidine and / or 5-hydroxymethylcytidine include, but are not limited to: pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, N4-acetylcytidine or one or more derivatives thereof. In certain embodiments, for example, when performing only a single transfection or when the cells being transfected are not particularly sensitive to transfection-associated toxicity or innate-immune signaling, the fractions of non-canonical nucleotides can be reduced. Reducing the fraction of non-canonical nucleotides can be beneficial, in part, because reducing the fraction of non-canonical nucleotides can reduce the cost of the nucleic acid. In certain situations, for example, when minimal immunogenicity of the nucleic acid is desired, the fractions of non-canonical nucleotides can be increased.

[0510] Note that alternative naming schemes exist for certain non-canonical nucleotides. For example, in certain situations, 5-methylpseudouridine can be referred to as “3-methylpseudouridine” or “N3-methylpseudouridine” or “1-methylpseudouridine” or “N1-methylpseudouridine”. Nucleotides that contain the prefix “amino” can refer to any nucleotide that contains a nitrogen atom bound to the atom at the stated position of the nucleotide, for example, 5-aminocytidine can refer to 5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly, nucleotides that contain the prefix “methyl” can refer to any nucleotide that contains a carbon atom bound to the atom at the stated position of the nucleotide, for example, 5-methylcytidine can refer to 5-methylcytidine, 5-ethylcytidine, and 5-hydroxymethylcytidine, nucleotides that contain the prefix “thio” can refer to any nucleotide that contains a sulfur atom bound to the atom at the given position of the nucleotide, and nucleotides that contain the prefix “hydroxy” can refer to any nucleotide that contains an oxygen atom bound to the atom at the given position of the nucleotide, for example, 5-hydroxyuridine can refer to 5-hydroxyuridine and uridine with a methyl group bound to an oxygen atom, wherein the oxygen atom is bound to the atom at the 5C position of the uridine.

[0511] In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the nucleic acid comprises one or more non-canonical nucleotide members of the 5 methylcytidine de-methylation pathway. In some embodiments, the nucleic acid comprises at least one of: 5 methylcytidine, 5 hydroxymethylcytidine, 5 formylcytidine, and 5 carboxycytidine or a derivative thereof. In some embodiments, the nucleic acid comprises at least one of: pseudouridine, 5 methylpseudouridine, 5 methyluridine, 5 methylcytidine, 5 hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof.

[0512] Certain non-canonical nucleotides can be incorporated more efficiently than other non-canonical nucleotides into RNA molecules by RNA polymerases that are commonly used for in vitro transcription, due in part to the tendency of these certain non-canonical nucleotides to participate in standard base-pairing interactions and base-stacking interactions, and to interact with the RNA polymerase in a manner similar to that in which the corresponding canonical nucleotide interacts with the RNA polymerase. As a result, certain nucleotide mixtures containing one or more non-canonical nucleotides can be beneficial in part because in vitro-transcription reactions containing these nucleotide mixtures can yield a large quantity of RNA. Certain embodiments are therefore directed to a nucleotide mixture containing one or more nucleotides that includes one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine. Nucleotide mixtures include, but are not limited to (numbers preceding each nucleotide indicate an exemplary fraction of the non-canonical nucleotide triphosphate in an in vitro-transcription reaction, for example, 0.2 pseudoisocytidine refers to a reaction containing adenosine-5′-triphosphate, guanosine-5′-triphosphate, uridine-5′-triphosphate, cytidine-5′-triphosphate, and pseudoisocytidine-5′-triphosphate, wherein pseudoisocytidine-5′-triphosphate is present in the reaction at an amount approximately equal to 0.2 times the total amount of pseudoisocytidine-5′-triphosphate+cytidine-5′-triphosphate that is present in the reaction, with amounts measured either on a molar or mass basis, and wherein more than one number preceding a nucleoside indicates a range of exemplary fractions): 1.0 pseudouridine, 0.1-0.8 2-thiouridine, 0.1-0.8 5-methyluridine, 0.2-1.0 5-hydroxyuridine, 0.2-1.0 5-methoxyuridine, 0.1-1.0 5-aminouridine, 0.1-1.0 4-thiouridine, 0.1-1.0 2-thiopseudouridine, 0.1-1.0 4-thiopseudouridine, 0.1-1.0 5-hydroxypseudouridine, 0.2-1 5-methylpseudouridine, 0.2-1.0 5-methoxypseudouridine, 0.1-1.0 5-aminopseudouridine, 0.2-1.0 2-thiocytidine, 0.1-0.8 pseudoisocytidine, 0.2-1.0 5-methylcytidine, 0.2-1.0 5-hydroxycytidine, 0.2-1.0 5-hydroxymethylcytidine, 0.2-1.0 5-methoxycytidine, 0.1-1.0 5-aminocytidine, 0.2-1.0 N4-methylcytidine, 0.2-1.0 5-methylpseudoisocytidine, 0.2-1.0 5-hydroxypseudoisocytidine, 0.2-1.0 5-aminopseudoisocytidine, 0.2-1.0 N4-methylpseudoisocytidine, 0.2-1.0 2-thiopseudoisocytidine, 0.2-1.0 7-deazaguanosine, 0.2-1.0 6-thioguanosine, 0.2-1.0 6-thio-7-deazaguanosine, 0.2-1.0 8-azaguanosine, 0.2-1.0 7-deaza-8-azaguanosine, 0.2-1.0 6-thio-8-azaguanosine, 0.1-0.5 7-deazaadenosine, and 0.1-0.5 N6-methyladenosine.

[0513] In some embodiments, the RNA comprising one or more non-canonical nucleotides composition or synthetic polynucleotide composition (e.g., which may be prepared by in vitro transcription) contains substantially or entirely the canonical nucleotide at positions having adenine or “A” in the genetic code. The term “substantially” in this context refers to at least 90%. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) 7-deazaguanosine at positions with “G” in the genetic code as well as the corresponding canonical nucleotide “G”, and the canonical and non-canonical nucleotide at positions with G may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three or four) of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine at positions with “C” in the genetic code as well as the canonical nucleotide “C”, and the canonical and non-canonical nucleotide at positions with C may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “C” are as described in the preceding paragraph. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three, or four) of 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine at positions with “U” in the genetic code as well as the canonical nucleotide “U”, and the canonical and non-canonical nucleotide at positions with “U” may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “U” are as described in the preceding paragraph.

[0514] In embodiments, combining certain non-canonical nucleotides can be beneficial in part because the contribution of non-canonical nucleotides to lowering the toxicity of RNA molecules can be additive. Certain embodiments are therefore directed to a nucleotide mixture, wherein the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, for example, the nucleotide mixture contains both pseudoisocytidine and 7-deazaguanosine or the nucleotide mixture contains both N4-methylcytidine and 7-deazaguanosine, etc. In one embodiment, the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, and each of the non-canonical nucleotides is present in the mixture at the fraction listed above, for example, the nucleotide mixture contains 0.1-0.8 pseudoisocytidine and 0.2-1.0 7-deazaguanosine or the nucleotide mixture contains 0.2-1.0 N4-methylcytidine and 0.2-1.0 7-deazaguanosine, etc.

[0515] In certain situations, for example, when it may not be necessary or desirable to maximize the yield of an in vitro-transcription reaction, nucleotide fractions other than those given above may be used. The exemplary fractions and ranges of fractions listed above relate to nucleotide-triphosphate solutions of typical purity (greater than 90% purity). Larger fractions of these and other nucleotides can be used by using nucleotide-triphosphate solutions of greater purity, for example, greater than about 95% purity or greater than about 98% purity or greater than about 99% purity or greater than about 99.5% purity, which can be achieved, for example, by purifying the nucleotide triphosphate solution using existing chemical-purification technologies such as high-pressure liquid chromatography (HPLC) or by other means. In one embodiment, nucleotides with multiple isomers are purified to enrich the desired isomer.

[0516] In some embodiments, the one or more non-canonical nucleotides avoids substantial cellular toxicity.

[0517] In some embodiments, the non-canonical nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine, optionally at an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-canonical nucleotides.

[0518] In some embodiments, at least about 50% of cytidine residues are non-canonical nucleotides, and which are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0519] In some embodiments, at least about 75% or at least about 90% of cytidine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0520] In some embodiments, at least about 20% of uridine, or at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0521] In some embodiments, at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0522] In some embodiments, at least about 10% of guanine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the synthetic RNA comprises no more than about 50% 7-deazaguanosine in place of guanosine residues. In some embodiments, the synthetic RNA does not comprise non-canonical nucleotides in place of adenosine residues.Methods of Treatment

[0523] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0524] In various embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a chimeric antigen receptor (CAR). In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the CAR inserts into the break. In these embodiments, the cell in the subject expresses the CAR.

[0525] In numerous embodiments, the method further comprises administering to the subject in need a synthetic mRNA encoding a gene-editing protein (e.g., a temperature-sensitive gene-editing protein) and a single-stranded or double-stranded repair template which encodes a cytokine. In some cases, the gene-editing protein creates a single-stranded break or a double-stranded break in the genomic DNA of a cell in the subject and the single-stranded or double-stranded repair template which encodes the cytokine inserts into the break. In these embodiments, the cell in the subject expresses or over expresses the cytokine.

[0526] When the synthetic mRNA and / or the repair template is administered to a subject, the synthetic mRNA and / or the repair is combined with a lipid system comprising a compound of Formula (IV).

[0527] In these embodiments, the cytotoxic lymphocyte targets and kills cancer cells and the isolated myeloid lineage cells kill cancer cells and / or promote cancer cell killing by cytotoxic lymphocytes.

[0528] In various embodiments, the present methods and compositions find use in methods of treating, preventing, or ameliorating a disease, disorder, and / or condition. For instance, in some embodiments, the described methods of in vivo delivery, including administration strategies, and formulations are used in a method of treatment. In some methods, the described methods reduce symptoms associated with a disease. In some embodiments, the methods eliminate the underlying cause of the disease. In some embodiments, the methods are used in the treatment of a disease requiring immunosuppression. In some embodiments, the methods reduce inflammation. In some embodiments, the methods reduce immune response.

[0529] In various embodiments, the present invention pertains to pharmaceutical compositions comprising the recombinantly engineered cells described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the present invention pertains to pharmaceutical compositions comprising the iPSC-derived cells of the lymphoid lineage, including cytotoxic lymphocytes, iPSC-derived cells of the myeloid lineage, e.g., monocytes which can be differentiated into functional M1 and M2 macrophages having enhanced cytokine secretion and tumor cell-killing activity, and / or synthetic RNA molecules encoding the gene-editing protein or expression cassettes for expressing a protein of interest, e.g., a CAR or for expressing or overexpressing a cytokine.

[0530] In embodiments, the disclosed composition is suitable for use in the treatment of amyotrophic lateral sclerosis (ALS), spinal cord injury, degenerative disc disease, coronary artery disease, acute myocardial infarction, alcoholic liver cirrhosis, hepatitis C virus (HCV)-induced cirrhosis, multiple sclerosis (MS), osteoarthritis (OA), osteoarthritis of the knee, kidney allograft, critical limb ischemia, ischemic cardiomyopathy, Crohn's disease, idiopathic pulmonary fibrosis, anal fistula, spinal cord injury, systemic lupus erythematosus (SLE), acute respiratory distress syndrome (ARDS), acute graft-versus-host disease (aGvHD), preterm bronchopulmonary dysplasia (BPD), autism nonischemic heart failure, and / or Type 2 diabetes mellitus.

[0531] In embodiments, the present methods relate to therapeutic use in autoimmune diseases or disorders. Examples of autoimmune diseases or disorders that may be treated or prevented by the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), irritable bowel disease (IBD), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis. Preferably autoimmune disorders that may be treated or prevented by the present compositions include rheumatoid arthritis, type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, and atopy.

[0532] In embodiments, the present methods relate to therapeutic use in degenerative diseases or disorders. A degenerative diseases or disorders is a disease in which the function or structure of the affected tissues or organs will progressively deteriorate overtime. Examples of degenerative diseases that can be treated or prevented with the present invention include Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Parkinson's Disease, Multiple system atrophy, Niemann Pick disease, Atherosclerosis, Progressive supranuclear palsy, Tay-Sachs Disease, Diabetes, Heart Disease, Keratoconus, Inflammatory Bowel Disease (IBD), Prostatitis, Osteoarthritis, Osteoporosis, Rheumatoid Arthritis, Huntington's Disease, Chronic traumatic encephalopathy, Epilepsy, Dementia, Renal failure, Multiple sclerosis, Malaria with CNS degeneration, Neuro-AIDS, Lysosomal storage diseases, Encephalitis of viral, bacterial or autoimmune origin.

[0533] In embodiments, the present methods relate to therapeutic use in a lung diseases or disorders.

[0534] In embodiments, the lung disease or disorder is a lung disease or disorder that would benefit therapeutically from suppression of immune responses in the lung. In some embodiments, inflammation is associated with the lung disease or disorder.

[0535] In some embodiments, the lung disease or disorder is selected from Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD), Common Cold, Croup, Cystic Fibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis, Influenza, Lung Cancer, Pandemic Flu, Pertussis, Pleurisy, Pneumonia, Pulmonary Embolism, Pulmonary Hypertension, Respiratory Syncytial Virus (RSV), Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS), and Tuberculosis.

[0536] In some embodiments, the lung disease or disorder is chronic obstructive pulmonary disease (COPD), reactive airway disease such as asthma, bronchiolitis, acute lung injury, lung allograft rejection (acute or chronic), pulmonary fibrosis, interstitial lung disease or hypersensitivity pneumonitis. In embodiments, the disease or disorder is an acute lung injury (ALI). In embodiments, the ALI is a pulmonary disorder that can be induced directly by inhalation of chemicals (chemical induced acute lung injury) or other means (e.g., infection) or can be induced indirectly by systemic injury (e.g., infection). Acute lung injury includes subcategories of respiratory distress syndromes including infant respiratory distress syndrome (IRDS), hyaline membrane disease (HMD), neonatal respiratory distress syndrome (NRDS), respiratory distress syndrome of newborn (RDSN), surfactant deficiency disorder (SDD), acute respiratory distress syndrome (ARDS), respiratory complication from systemic inflammatory response syndrome (SIRS), or severe acute respiratory syndrome (SARS).

[0537] In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms associated with a viral infection.

[0538] In embodiments, the composition is suitable for use in the treatment of an infectious disease, optionally selected from an infection with a pathogen, optionally selected from a bacterium, virus, fungus, or parasite.

[0539] In embodiments, the pathogen is a virus. In embodiments, the virus is: (a) an influenza virus, optionally selected from Type A, Type B, Type C, and Type D influenza viruses, or (b) a member of the Coronaviridae family, optionally selected from (i) a betacoronavirus, optionally selected from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43 or (ii) an alphacoronavirus, optionally selected from HCoV-NL63 and HCoV-229E.

[0540] In embodiments, the virus is SARS-CoV-2. In embodiments, the virus is SARS-CoV-2, which has caused COVID-19. In embodiments, the COVID-19 is characterized by one or more of fever, cough, shortness of breath, diarrhea, upper respiratory symptoms, lower respiratory symptoms, pneumonia, and respiratory distress.

[0541] In some embodiments, the composition is suitable for use in the treatment of an infection, wherein the infection is a coronavirus infection. In some embodiments, the coronavirus infection is one or more of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E. In various embodiments, the coronavirus infection is SARS or COVID-19. In further embodiments, the subject is infected by SARS-CoV-2.

[0542] In embodiments, the therapy prevents or mitigates development of acute respiratory distress syndrome (ARDS) in a patient when administered. In embodiments, the therapy improves oxygenation in a patient when administered. In embodiments, the therapy improves systemic blood pressure oxygenation in a patient when administered, e.g., reducing or mitigating shock, e.g., requiring less pressor support. In embodiments, the therapy improves lung and / or alveolar permeability in a patient when administered.

[0543] In embodiments, the therapy prevents or mitigates a transition from respiratory distress to cytokine imbalance in a patient when administered. In embodiments, the therapy reverses or prevents a cytokine storm in a patient when administered. In embodiments, the therapy reverses or prevents a cytokine storm in the lungs or systemically in a patient when administered. In embodiments, the cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis.

[0544] In embodiments, the therapy reverses or prevents excessive production of one or more inflammatory cytokines in a patient when administered. In embodiments, the inflammatory cytokine is one or more of IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

[0545] In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms associated with a coronavirus infection.

[0546] Coronaviruses (CoVs) are members of the family Coronaviridae, including betacoronavirus and alphacoronavirus—respiratory pathogens that have relatively recently become known to invade humans. The Coronaviridae family includes such betacoronavirus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43. Alphacoronavirus includes, e.g., HCoV-NL63 and HCoV-229E. In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms of infection with any of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43. Alphacoronavirus includes, e.g., HCoV-NL63 and HCoV-229E.

[0547] Without wishing to be bound by theory, coronaviruses invade cells through utilization of their “spike” surface glycoprotein that is responsible for viral recognition of Angiotensin Converting Enzyme 2 (ACE2), a transmembrane receptor on mammalian hosts that facilitate viral entrance into host cells. (Zhou et al., A pneumonia outbreak associated with anew coronavirus of probable bat origin, Nature 2020).

[0548] Symptoms associated with coronavirus infections include, but are not limited to, fever, tiredness, dry cough, aches and pains, shortness of breath and other breathing difficulties, diarrhea, upper respiratory symptoms (e.g., sneezing, runny nose, nasal congestion, cough, sore throat), and / or pneumonia. In embodiments, the present compositions and methods are useful in treating or mitigating any of these symptoms.

[0549] In embodiments, the present invention relates to the therapeutic use of the present cells for the treatment of one or more symptoms of infection with SARS-CoV-2, including Coronavirus infection 2019 (COVID-19), caused by SARS-CoV-2 (e.g., 2019-nCoV).

[0550] In some settings, including subjects afflicted with coronavirus infections, it is possible that the morbidity and mortality of pulmonary viral infection is related to an exaggerated or overwhelming inflammatory response. In varying clinical circumstances this response can be described as “cytokine response syndrome”, “cytokine storm”, or “secondary hemophagocytic lymphohistiocytosis” (sHLH). In embodiments, the present compositions and methods are useful in treating or mitigating any of these exaggerated or overwhelming inflammatory responses. Collectively it is surmised that these highly proinflammatory states can lead to death due to pulmonary collapse such as acute respiratory distress syndrome (ARDS) or systemic, multi-organ failure affecting organs such as liver, kidney, heart and brain. In embodiments, the present cells treat or mitigate a “cytokine response syndrome”, “cytokine storm”, or “secondary hemophagocytic lymphohistiocytosis” (sHLH).

[0551] In embodiments, COVID-19 is characterized, in part, by elevation of Interleukin-2 (IL-2), Interleukin-7 (IL-7), granulocyte colony stimulating factor (GCSF), interferon-gamma inducible protein 10, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP1a), and tumor necrosis factor-alpha (TNFα). In embodiments, the present compositions and methods are useful in treating or mitigating increases of any of these factors.

[0552] In embodiments, the present cells prevent a COVID-19 patient from having a disease that develops from respiratory distress to cytokine storm.

[0553] In embodiments, the present cells treat or mitigate ARDS.

[0554] In some embodiments, a cytokine storm is associated with COVID-19 and is treated or mitigated via a method comprising administering to a subject in need thereof an effective amount of cells effective for the treatment of a coronavirus infection and / or a cytokine storm associated with a coronavirus infection, wherein the subject has abnormal (e.g. increased or decreased) expression or activity of one or more of IL-6, IL-1, TNF, interferon-γ, CXCL10, CCL7, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, CCL2 / MCP-1, CCL5 / RANTES, CCL7 / MCP-3, MCP-2, tumor necrosis factor-alpha (TNFα), interferon-γ (IFNγ), CXCL10, CXC3, Granulocyte colony stimulatory factor (GCSF), Macrophage inflammatory protein 1 alpha (MIP-1a), IL-22, and Interferon gamma induced protein 10 (IP-10).

[0555] In some embodiments, the subject has a modulated (e.g. decreased or increased) expression or activity of one or more of IL-6, IL-1, TNF, interferon-γ, CXCL10, CCL7, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, CCL2 / MCP-1, CCL5 / RANTES, CCL7 / MCP-3, MCP-2, tumor necrosis factor-alpha (TNFα), interferon-γ (IFNγ), CXCL10, CXC3, Granulocyte colony stimulatory factor (GCSF), Macrophage inflammatory protein 1 alpha (MIP-1a), IL-22, and Interferon gamma induced protein 10 (IP-10).

[0556] In embodiments, the disease / indication is associated with one or more cancers. The one or more cancers may comprise: adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann Syndrome, bile duct caner (Cholangiocarcinoma), Birt-Hogg Dube Syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, breast cancer (inflammatory), breast cancer (metastatic), breast cancer in men, carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma tumor, ependymoma, esophageal cancer, Ewing Sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor GIST, germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, leukemia—acute lymphoblastic—ALL, leukemia, acute lymphocytic—ALL, leukemia—acute myeloid—ALL, leukemia—acute myeloid—AML, leukemia—B-cell prolymphocytic leukemia and hairy cell leukemia, leukemia—chronic lymphocytic—CLL, leukemia—chronic myeloid—CML, leukemia—chronic t-cell lymphocytic, leukemia—eosinophilic, Li-Fraumeni Syndrome, liver cancer, lung cancer—non-small cell, lung cancer—small cell, lymphoma—Hodgkin, lymphoma—Non-Hodgkin, Lynch Syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH / MYH—associated polyposis, myelodysplastic syndromes—MDS, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers Syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma—Kaposi, sarcomas of specific organs, sarcomas—soft tissue, skin cancer (non-melanoma), skin cancer (melanoma), small bowel cancer, stomach cancer, testicular cancer, thyoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, unknown primary, uterine cancer, vaginal cancer, Von Hippel-Lindau Syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner Syndrome, Wilms tumor, and xeroderma pigmentosum.

[0557] In an aspect, the present disclosure provides a method for treating a cancer in a patient in need thereof. The method comprising administering to the cancer patient a therapeutically-effective amounts of any herein-disclosed cytotoxic lymphocyte.

[0558] An aspect of the present disclosure is a method for killing a cancer cell. The method comprising steps of: (1) obtaining a herein-disclosed cytotoxic lymphocyte and (2) contacting cytotoxic lymphocyte with the cancer. In some cases, the cancer cell is in vivo.

[0559] Yet another aspect of the present disclosure is a method for treating a cancer patient in need thereof. The method comprising a step of administering to the cancer patient a therapeutically-effective amounts of a herein-disclosed cytotoxic lymphocyte.

[0560] In aspects, the present disclosure provides a method of treating cancer, comprising: (a) obtaining an isolated cytotoxic lymphocyte comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene; and (b) administering the isolated cytotoxic lymphocyte to a subject in need thereof.

[0561] In some cases, the lymphoid lineage cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell.

[0562] In some cases, the myeloid lineage cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0563] In embodiments, the cytotoxic lymphocyte is an NK cell.

[0564] In embodiments, the cancer is a blood cancer. In embodiments, the cancer is a solid tumor. In embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), and Meigs' syndrome.

[0565] The cytotoxic lymphocyte of the present disclosure may be administered systemically (e.g., via a vein or artery) or may be introduced into a tumor or in the vicinity of the tumor.Administration and Formulations

[0566] In some embodiments, the present disclosure relates to compositions described herein in the form of a pharmaceutical composition.

[0567] An aspect of the present disclosure is a method for treating a cancer. The method comprising administering to a subject in need a therapeutically-effective amount of a first pharmaceutical composition comprising one or both of a population of isolated lymphoid lineage cells and a population of isolated myeloid lineage cells.

[0568] In embodiments, the first pharmaceutical composition comprises the population of isolated lymphoid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated myeloid lineage cells.

[0569] In some embodiments, the first pharmaceutical composition comprises the population of isolated myeloid lineage cells and wherein the subject in need is administered a therapeutically-effective amount of a second pharmaceutical composition comprising a population of isolated lymphoid lineage cells. In some cases, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or sequentially. The first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the first pharmaceutical composition administered before the second pharmaceutical composition or the first pharmaceutical composition and the second pharmaceutical composition may be administered sequentially with the second pharmaceutical composition administered before the first pharmaceutical composition.

[0570] In various embodiments, the first pharmaceutical composition comprises both the population of isolated lymphoid lineage cells and the population of isolated myeloid lineage cells.

[0571] In various embodiments, the present invention pertains to pharmaceutical compositions comprising the recombinantly engineered cells described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the present invention pertains to pharmaceutical compositions comprising the iPSC-derived cells of the lymphoid lineage, including cytotoxic lymphocytes, iPSC-derived cells of the myeloid lineage, e.g., monocytes which can be differentiated into functional M1 and M2 macrophages having enhanced cytokine secretion and tumor cell-killing activity, and / or synthetic RNA molecules encoding the gene-editing protein or expression cassettes for expressing a protein of interest, e.g., a CAR or for expressing or overexpressing a cytokine.

[0572] Therapeutic treatments comprise the use of one or more routes of administration and of one or more formulations that are designed to achieve a therapeutic effect at an effective dose, while minimizing toxicity to the subject to which treatment is administered. Illustrative formulations / compositions of the present disclosure include engineered cells along with a suitable delivery reagent, e.g., a liquid carrier.

[0573] In various embodiments, the effective dose is an amount that substantially avoids cell toxicity in vivo. In various embodiments, the effective dose is an amount that substantially avoids an immune reaction in a human subject. For example, the immune reaction may be an immune response mediated by the innate immune system. Immune response can be monitored using markers known in the art (e.g., cytokines, interferons, TLRs). In some embodiments, the effective dose obviates the need for treatment of the human subject with immune suppressants agents (e.g., B18R) used to moderate the residual toxicity.

[0574] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective, as described herein. The formulations may easily be administered in a variety of dosage forms such as injectable solutions and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered, and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art. Pharmaceutical preparations may additionally comprise delivery reagents (a.k.a. “vehicles”, a.k.a. “delivery vehicles”) and / or excipients. Pharmaceutically acceptable delivery reagents, excipients, and methods of preparation and use thereof, including methods for preparing and administering pharmaceutical preparations to patients (a.k.a. “subjects”) are well known in the art, and are set forth in numerous publications, including, for example, in US Patent Appl. Pub. No. US 2008 / 0213377, the entirety of which is incorporated herein by reference.

[0575] The present pharmaceutical compositions can comprise excipients, including liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0576] In embodiments, the composition is formulated for one or more of intrathecal, intra-lesional, intra-coronary, intravenous (IV), intra-articular, intramuscular, and intra-endobronchial administration and administration via intrapancreatic endovascular injection, intra-nucleus pulposus, lumbar puncture, intra-myocardium, transendocardium, intra-fistula tract, intermedullary space, intradural space and leg injection.

[0577] In embodiments, the composition is formulated for infusion. In some embodiments, the composition is formulated for infusion, wherein the composition is delivered to the bloodstream of a subject or patient through a needle in a vein of the subject or patient through a peripheral line, a central line, a tunneled line, an implantable port, and / or a catheter. In some embodiments, the subject or patient may also receive supportive medications or treatments, such as hydration, by infusion. In some embodiments, the composition is formulated for intravenous infusion. In some embodiments, the infusion is continuous infusion, secondary intravenous therapy (IV), and / or IV push. In some embodiments, the infusion of the composition may be administered through the use of equipment selected from one or more of an infusion pump, hypodermic needle, drip chamber, peripheral cannula, and pressure bag.

[0578] In embodiments, the method of treating a subject comprises administering a cell of the present disclosure to a subject in need thereof. In embodiments, the cell is formulated for therapeutic use. In embodiments, the cell is suitable for administration to a human subject. In embodiments, the method is conducted in vivo.

[0579] In numerous embodiments, the administering is intravenous, intraarterial, intratumoral, or injected in the vicinity of a tumor.

[0580] In embodiments when a synthetic RNA molecule encoding a temperature-sensitive gene-editing protein is administered, the method comprises reducing the body temperature of a subject, optionally via whole-body hypothermia. In embodiments, the body temperature of the subject is reduced by from about 0.5° C. to about 1° C. In embodiments, the body temperature of the subject is reduced by from about 1° C. to about 1.5° C. In embodiments, the body temperature of the subject is reduced by from about 1.5° C. to about 2° C. In embodiments, the body temperature of the subject is reduced by from about 2° C. to about 2.5° C. In embodiments, the body temperature of the subject is reduced by from about 2.5° C. to about 3° C. In embodiments, the body temperature of the subject is reduced by from about 3° C. to about 3.5° C. In embodiments, the body temperature of the subject is reduced by from about 3.5° C. to about 4° C. In embodiments, the body temperature of the subject is reduced by from about 4° C. to about 4.5° C. In embodiments, the body temperature of the subject is reduced by from about 4.5° C. to about 5° C. In embodiments, the body temperature of the subject is reduced by from about 5° C. to about 5.5° C. In embodiments, the body temperature of the subject is reduced by from about 5.5° C. to about 6° C. In embodiments, the body temperature of the subject is reduced by from about 6° C. to about 6.5° C. In embodiments, the body temperature of the subject is reduced by from about 6.5° C. to about 7° C. In embodiments, the body temperature of the subject is reduced by from about 7° C. to about 7.5° C. In embodiments, the body temperature of the subject is reduced by from about 7.5° C. to about 8° C. In embodiments, the body temperature of the subject is reduced by from about 8° C. to about 8.5° C. In embodiments, the body temperature of the subject is reduced by from about 8.5° C. to about 9° C. In embodiments, the body temperature of the subject is reduced by from about 9° C. to about 9.5° C. In embodiments, the body temperature of the subject is reduced by from about 9.5° C. to about 10° C. In embodiments, the body temperature of the subject is reduced by from about 10° C. to about 10.5° C. In embodiments, the body temperature of the subject is reduced by from about 10.5° C. to about 11° C. In embodiments, the body temperature of the subject is reduced by from about 11° C. to about 11.5° C. In some embodiments, the reducing the body temperature of the subject is performed for a specific amount of time. In some embodiments, the specific amount of time is from about 15 minutes to about 30 minutes. In some embodiments, the specific amount of time is from about 30 minutes to about 45 minutes. In some embodiments, the specific amount of time is from about 45 minutes to about 60 minutes. In embodiments, the specific amount of time is from about 1 hour to about 1.5 hours. In embodiments, the specific amount of time is from about 1.5 hours to about 2 hours. In embodiments, the specific amount of time is from about 2 hours to about 2.5 hours. In embodiments, the specific amount of time is from about 2.5 hours to about 3 hours. In embodiments, the specific amount of time is from about 3 hours to about 3.5 hours. In embodiments, the specific amount of time is from about 3.5 hours to about 4 hours. In embodiments, the specific amount of time is from about 4 hours to about 4.5 hours. In embodiments, the specific amount of time is from about 4.5 hours to about 5 hours. In embodiments, the specific amount of time is from about 5 hours to about 5.5 hours. In embodiments, the specific amount of time is from about 5.5 hours to about 6 hours. In embodiments, the specific amount of time is from about 6 hours to about 6.5 hours.

[0581] In embodiments when a synthetic RNA molecule encoding a temperature-sensitive gene-editing protein is administered, the method comprises applying one or more cooling elements to a cell or tissue in vivo to reduce temperature, the cooling element optionally being a cryocompression device. In embodiments, the temperature is reduced by from about 0.5° C. to about 1° C. In embodiments, the temperature is reduced by from about 1° C. to about 1.5° C. In embodiments, the temperature is reduced by from about 1.5° C. to about 2° C. In embodiments, the temperature is reduced by from about 2° C. to about 2.5° C. In embodiments, the temperature is reduced by from about 2.5° C. to about 3° C. In embodiments, the temperature is reduced by from about 3° C. to about 3.5° C. In embodiments, the temperature is reduced by from about 3.5° C. to about 4° C. In embodiments, the temperature is reduced by from about 4° C. to about 4.5° C. In embodiments, the temperature is reduced by from about 4.5° C. to about 5° C. In embodiments, the temperature is reduced by from about 5° C. to about 5.5° C. In embodiments, the temperature is reduced by from about 5.5° C. to about 6° C. In embodiments, the temperature is reduced by from about 6° C. to about 6.5° C. In embodiments, the temperature is reduced by from about 6.5° C. to about 7° C. In embodiments, the temperature is reduced by from about 7° C. to about 7.5° C. In embodiments, the temperature is reduced by from about 7.5° C. to about 8° C. In embodiments, the temperature is reduced by from about 8° C. to about 8.5° C. In embodiments, the temperature is reduced by from about 8.5° C. to about 9° C. In embodiments, the temperature is reduced by from about 9° C. to about 9.5° C. In embodiments, the temperature is reduced by from about 9.5° C. to about 10° C. In embodiments, the temperature is reduced by from about 10° C. to about 10.5° C. In embodiments, the temperature is reduced by from about 10.5° C. to about 11° C. In embodiments, the temperature is reduced by from about 11° C. to about 11.5° C.

[0582] In embodiments when a synthetic RNA molecule encoding a temperature-sensitive gene-editing protein is administered, the applying one or more cooling elements to a cell or tissue in vivo to reduce temperature is performed for a specific amount of time. In some embodiments, the specific amount of time is from about 15 minutes to about 30 minutes. In some embodiments, the specific amount of time is from about 30 minutes to about 45 minutes. In some embodiments, the specific amount of time is from about 45 minutes to about 60 minutes. In some embodiments, the specific amount of time is from about 1 hour to about 1.5 hours. In some embodiments, the specific amount of time is from about 1.5 hours to about 2 hours. In some embodiments, the specific amount of time is from about 2 hours to about 2.5 hours. In some embodiments, the specific amount of time is from about 2.5 hours to about 3 hours. In some embodiments, the specific amount of time is from about 3 hours to about 3.5 hours. In some embodiments, the specific amount of time is from about 3.5 hours to about 4 hours. In some embodiments, the specific amount of time is from about 4 hours to about 4.5 hours. In some embodiments, the specific amount of time is from about 4.5 hours to about 5 hours. In some embodiments, the specific amount of time is from about 5 hours to about 5.5 hours. In some embodiments, the specific amount of time is from about 5.5 hours to about 6 hours. In some embodiments, the specific amount of time is from about 6 hours to about 6.5 hours.

[0583] Further description of temperature-sensitive cell administration is found in WO2021 / 231549. The entire contents of which are incorporated by reference in their entirety.

[0584] In some embodiments, the present invention relates to one or more administration techniques described in U.S. Pat. Nos. 5,711,964; 5,891,468; 6,316,260; 6,413,544; 6,770,291; and 7,390,780, the entire contents of which are hereby incorporated by reference in their entireties.Lipids Cell Contacting Transfection

[0585] In embodiments, the present invention relates delivery of the present synthetic RNA molecules via a lipid. In some embodiments, mRNAs encoding a gene-editing protein and / or a reprogramming factor are delivered via a lipid.

[0586] In embodiments, the lipid is a compound of Formula (I)wherein: Q1, Q2, Q3, and Q4 are independently an atom or group capable of adopting a positive charge;

[0588] A1 and A2 are independently null, H, or optionally substituted C1-C6 alkyl;

[0589] L1, L2, and L3 are independently null, a bond, (C1-C20)alkanediyl, (halo)(C1-C20)alkanediyl, (hydroxy)(C1-C20)alkanediyl, (alkoxy)(C1-C20)alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, or any combination of the aforementioned optionally linked by one or more of an ether, an ester, an anhydride, an amide, a carbamate, a secondary amine, a tertiary amine, a quaternary ammonium, a thioether, a urea, a carbonyl, or an imine;

[0590] R1, R2, R3, R4, R5, R6, R7, and R8 are independently null, H, (C1-C60)alkyl, (halo)(C1-C60)alkyl, (hydroxy)(C1-C60)alkyl, (alkoxy)(C1-C60)alkyl, (C2-C60)alkenyl, (halo)(C2-C60)alkenyl, (hydroxy)(C2-C60)alkenyl, (alkoxy)(C2-C60)alkenyl, (C2-C60)alkynyl, (halo)(C2-C60)alkynyl, (hydroxy)(C2-C60)alkynyl, (alkoxy)(C2-C60)alkynyl, wherein at least one of R1, R2, R3, R4, R5, R6, R7, and R8 comprises at least two unsaturated bonds; and x, y, and z are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0591] In embodiments, the lipid is a compound of Formula (II):wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, and R28 are independently H, halo, OH, (C1-C6)alkyl, (halo)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclo; and

[0593] i, j, k, m, s, and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0594] In embodiments, the lipid is a compound of Formula (III):wherein L4, L5, L6, and L7 are independently a bond, (C1-C20)alkanediyl, (halo)(C1-C20)alkanediyl, (hydroxy)(C1-C20)alkanediyl, (alkoxy)(C1-C20)alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, —(CH2)v1—C(O)—, —((CH2)v1—O)v2—, or —((CH2)v1—C(O)—O)v2—;

[0596] R29, R30, R31, R32, R33, R34, and R35 are independently H, (C1-C60)alkyl, (halo)(C1-C60)alkyl, (hydroxy)(C1-C60)alkyl, (alkoxy)(C1-C60)alkyl, (C2-C60)alkenyl, (halo)(C2-C60)alkenyl, (hydroxy)(C2-C60)alkenyl, (alkoxy)(C2-C60)alkenyl, (C2-C60)alkynyl, (halo)(C2-C60)alkynyl, (hydroxy)(C2-C60)alkynyl, (alkoxy)(C2-C60)alkynyl, wherein at least one of R29, R30, R31, R32, R33, R34, and R35 comprises at least two unsaturated bonds;

[0597] v, v1 and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0598] In embodiments, the lipid is a compound of Formula (IV):wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0600] In embodiments, the lipid is a compound of Formula (V):

[0601] In embodiments, the lipid is a compound of Formula (VI):

[0602] In embodiments, the lipid is a compound of Fo...

Examples

example 1

Cell Preparation Methods and Results

[1094]FIG. 1A shows schematic of cellular production methods used in this Example. mRNA-based cellular reprogramming (fibroblast to iPS cell) and gene-editing (beta-2-microglobulin (B2M) knockout) were employed. Further, edited cells were differentiated into cytotoxic lymphoid cells. FIG. 1B illustrates the differentiated cytotoxic lymphoid cells killing cancer cells.

[1095]Fibroblast cells were obtained from a human subject and reprogrammed to iPS cells using an mRNA-based reprogramming.

[1096]Efficient targeting of defined loci in iPSCs using messenger RNA (mRNA)-encoded gene-editing endonucleases comprising DNA-binding domains containing novel linker region was undertaken (e.g., a gene-editing protein comprising a DNA binding domain having at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (SEQ ID NO: 65; the “*RVD*” corresponds to the dinucleotide “xy” of SEQ ID NO: 22). Exon 3 of B2M, a key component of MHC class I molecules was tar...

example 2

Cells Characterization Methods and Results

[1114]Phenotypical and functional characterization assays were used to evaluate cells of Example 1. Phenotypical characterization used flow cytometry staining of surface markers, specifically CD56 / CD16 (e.g., gated on CD56). CD56 / NKG2D, CD56 / CD45, CD56 / CD3, CD56 / CD244, CD56 / CD94 / NKG2A, CD56 / NKp46, CD56 / NKp44, CD56 / KIRs, CD56 / TRAIL, and CD56 / FASL were also assessed (e.g., gated on CD56). See, FIG. 10A to FIG. 10C and the below tables:

[1115]Data in this first table characterize PBMC-Isolated NK Cells vs. iPSC-Derived NK Cells from suspension round 1:

WTB2M− / −IsolatedPopulationPopulationPopulationPopulationNK Cells1212% of25.6%74.4%32.9%67.1%PopulationCD5677.5%82.1%22.3%84.9%68.2%CD1616.3%2.06%0.25%8.23%21.3%CD328.0%76.1%20.0%61.5%22.9%CD4597.8%99.1%99.4%99.8%99.8%NKG2D9.17%20.9%59.3%17.0%43.8%

[1116]Data in this second table characterize iPSC-Derived NK Cells—AggreWell™ vs. iPSC-Derived NK Cells from suspension round 2

AggreWell ™SuspensionWTB2M−...

example 3

B2M-HLA-E Insertion

[1128]In this example, repair template (the B2M-HLA-E repair template) comprising the B2M coding sequence, and the HLA-E (Major Histocompatibility Complex, Class I, E) coding sequence was inserted into a beta-2-microglobulin (B2M) edit. Here, iPSCs having their B2M gene edited, as disclosed herein, are contacted with a repair template comprising the coding sequence for HLA-E. Alternately, un-edited iPSCs are contacted with the gene-editing components to edit the B2M gene along with a repair template comprising the coding sequence for HLA-E. In both cases, the resulting cell (either as in iPSC or when differentiated into a cytotoxic lymphocyte of the lymphoid or differentiated into cells of the myeloid lineage, e.g., macrophages, or mesenchymal stromal / stem cells, or hematopoietic stem cells) will have an edited B2M gene and will express, in its place, HLA-E.

[1129]As shown in FIG. 11A the B2M signal peptide sequence (B2M_sp), which is contained entirely within Exon...

Claims

1. -59. (canceled)60. A method of engineering a cell, the method comprising(a) contacting a cell with a synthetic RNA encoding a gene-editing protein, wherein the gene-editing protein generates a break in a target site of a beta-2-microglobulin (B2M) gene, and(b) delivering to the cell a repair template comprising a nucleic acid sequence, wherein the nucleic acid sequence encodes an HLA-E polypeptide, wherein the nucleic acid sequence is inserted at the break in the target site of the B2M gene,thereby generating an engineered cell.

61. The method of claim 60, wherein the nucleic acid sequence encodes a fusion polypeptide comprising the HLA-E polypeptide and a B2M polypeptide.

62. The method of claim 61, wherein the repair template comprises a nucleic acid sequence encoding a signal peptide.

63. The method of claim 60, wherein the break is a double strand break.

64. The method of claim 60, wherein the target site of the B2M gene comprises a sequence of SEQ ID NO: 12.

65. The method of claim 60, wherein the target site of the B2M gene is in Exon 1 of the B2M gene.

66. The method of claim 60, wherein the cell is an immune cell selected from the group consisting of a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a T cell, or a macrophage.

67. The method of claim 60, wherein the cell is a stem cell.

68. The method of claim 67, wherein the method further comprises generating the stem cell from a somatic cell.

69. The method of claim 60, wherein the method results in reduced surface expression of HLA-A, HLA-B, or HLA-C by the engineered cell.

70. The method of claim 60, wherein the method further comprises (c) contacting the cell with a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

71. The method of claim 60, wherein the cell is further engineered to express a cytokine.

72. The method of claim 71, wherein the cytokine is selected from the group consisting of Interleukin (IL)-2, IL-15, IL-12, IL-7, IL-10, and IL-21.

73. The method of claim 60, wherein the gene-editing protein is selected from the group consisting of a transcription activator-like (TAL) effector nuclease, RiboSlice, a zinc-finger nuclease, a meganuclease, a nickase, or a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein.

74. The method of claim 60, wherein the synthetic RNA comprises one or more non-canonical nucleotides.

75. The method of claim 74, wherein the one or more non-canonical nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

76. The method of claim 60, wherein the synthetic RNA, the repair template, or both the synthetic RNA and the repair template are delivered to the cell in combination with a lipid system.

77. The method of claim 76, wherein the lipid system comprises a compound of Formula (IV):wherein n is an integer from 1 to 15.

78. The method of claim 77, wherein n is 4.

79. An engineered cell comprising a disruption in a B2M gene, wherein the engineered cell expresses a fusion protein comprising a fragment of a B2M polypeptide and a HLA-E polypeptide.

Images